pcr detection of hop fungal pathogens - Bayerische Landesanstalt
Detection of plant pathogens using non pcr based techniques
Transcript of Detection of plant pathogens using non pcr based techniques
NON-PCR BASED MOLECULAR
APPROACHES FOR DETECTION AND
IDENTIFICATION OF PLANT PATHOGENSDOCTORAL SEMINAR II
PUJA PANDEY41124
1
INTRODUCTION
Plant Pathology
Molecular Plant Pathology
2
bull Visual observations
bull Cultural
bull Microscopy
CONVENTIONAL TECHNIQUES
3
VISUAL OBSERVATION
SIGNS AND SYMPTOMS
Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans
Citrus canker
4
Problem with biotic and abiotic factors
5
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
INTRODUCTION
Plant Pathology
Molecular Plant Pathology
2
bull Visual observations
bull Cultural
bull Microscopy
CONVENTIONAL TECHNIQUES
3
VISUAL OBSERVATION
SIGNS AND SYMPTOMS
Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans
Citrus canker
4
Problem with biotic and abiotic factors
5
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
bull Visual observations
bull Cultural
bull Microscopy
CONVENTIONAL TECHNIQUES
3
VISUAL OBSERVATION
SIGNS AND SYMPTOMS
Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans
Citrus canker
4
Problem with biotic and abiotic factors
5
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
VISUAL OBSERVATION
SIGNS AND SYMPTOMS
Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans
Citrus canker
4
Problem with biotic and abiotic factors
5
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Problem with biotic and abiotic factors
5
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
CULTURAL LABORATORY TECHNIQUES
Bacterial ooze
Cultures of Ralstonia solanacearum
6
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Fungal cultureshellip
Alternaria culture Fusarium culture
7
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
MICROSCOPY An Aid To
The Eyes
Microscope
Stereoscopic Microscope
Fluorescence Microscope
Light Microscope
Bright Field Dark FieldPhase
ContrastConfocal
Electron Microscope
Scanning electron
Microscope
Transmission electron
Microscope
8
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Types of Microscope
1) Stereoscopic microscope
Function Visible light to illuminate the surface
of a sample (2000x ) without disrupting them
2) Compound microscope (light)
Function Visible light to illuminate a thin
section of sample cells and tissues
3) Confocal laser scanning fluorescence
microscope
Function Thin lsquoslicesrsquo in a sample while
keeping sample intact
Specifically at parts of a cell (such as
individual proteins) by labelling them with
fluorescence
9
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Scanning Electron Microscope
Function Surface of objects at high resolution
(3D image - 500 000x )
Principle Beam of electrons being knocked off
the surface of the sample and then picked up by a
detector
Why we use SEM
The SEM probably gives the best depth of field
out of any microscope
10
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Transmission Electron Microscope
Function very thin cross-section of an
object (cell) internal structure of objects
high resolution (500 000x )
Principle Electrons pass through the
sample and some are deflected and some
pass right through and that forms our
image which is focussed on objective
lens
11
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Microscopy Techniques Applied to the Study of Phytoplasma
Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali
12
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Light Microscopy
Dienesrsquostain was first developed
as a specific stain for animal
mycoplasma colonies
Phloem tissues of stems infected
by phytoplasmas stained dark
blue while xylem was turquoise
and cortex light blue
Musetti and Favali 2004
13
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Fluorescence Microscopy
DAPI staining of hand cut
sections of healthy
Antibody plus fluorochrome
such as fluorescein
isothiocyanate (FITC) and
stained
Phytoplasma-infected
the fluorescent bright spots
visible at phloem level
Musetti and Favali 200414
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Transmission electron microscopy (TEM)
Phytoplasmas in the phloem
cells of Catharanthus roseus
Musetti and Favali 200415
Phytoplasmas in the phloem of
apple tissues
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Immuno-electron microscopy (IEM) of thin sections
Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary
monoclonal antibody and gold Labelledsecondary antibody
Gold partcle (15 nm) few
particles are visible on
phytoplasma membrane
Gold partcle (5 nm) particles are
well distributed over the periphery
of the phytoplasmas
Musetti and Favali 200416
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
High resolution autoradiography
Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3
hours labelling with thymidine-3H The silver grains were seen on the
dividing phytoplasmas
17
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Staining Technique For Histopathological Tests
bull Staining is an auxiliary technique used in microscopy to
enhance contrast in the microscopic image
eg Crystal violet stains only Gram positive bacteria
18
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
19
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Histopathological analysis of infected tissues
20
Valencia sweet
oranges infected
with
Colletotrichum
acutatum
Valenciasweet orange
fruits infected
with Guignardia
citricarpa
Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
21
Limitations Faced due to conventional techniques
Latent infection eg Potato ring rot
Misleading infection eg Black lesions (Alternaria) and bacterial
blight of carrot (Xanthomonas)
Co-infection Alteration of symptoms
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
SEROLOGICAL METHODS ndash De Vorac
Antigen Antibody
Antigen Antibody
Positive
Result 22
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Antigen
A molecule usually a protein when it is injected into a
warm blooded animal produces antibody (immune
response)
Antibody
A molecule produced in a warm
blooded serum of animal in
response to the stimulus antigens
Antibodies are immune system-
related proteins called
immunoglobulin
23
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Variable region composed of 110-
130 amino acids give the antibody
its specificity for binding antigen
Variable region includes the ends of
the light and heavy chains
Constant region determines the
mechanism used to destroy antigen
Structure of Antibody
24
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODY
Composed of a variety of antibody
Have multiple epitopes
Antibody derived from a single
clone and specific for a single
epitope
Consist of single type of antibody
Produced by hybridoma technique
Small quantity of antigen is enough
for development
25
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Production of Mabs by Hybridoma Technique
26
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Antigen antibody based technique
Direct test
Precipitation test
1 Tube precipitation
2 Ring precipitation
Micro Precipitation test
Agglutination tests
1 Chloroplast agglutination
2 Latex agglutination
Gel diffusion test
Immuno-electrophoresis
Indirect test
ELISA test
1 Direct ELISA (DAS ELISA)
2 Indirect (DAC ELISA)
3 DIBA ELISA
4 Lateral flow device
Immunofluorescence
Immuno Sorbent Electron Microsopy(ISEM)
Flow cytometry
27
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Tube precipitation test
Widely used
Reactants diluted in 85gl
NaCl followed by
incubation at 37degC in water
bath
Observations
If elongated virus particle -
floccular
If spherical virus particle -
granular
Precipitation test
28
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Done on a micro-scale to
economize on antiserum
Drop of dilution mixture (antiserum
amp virus suspension) are mixed at
bottom of a Petri plate
The precipitates produced are
observed with a microscope with
dark-ground illumination
Precipitation varies depending on
the ratio of concentration of antigen
and antibody
Micro precipitition test
29
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Latex agglutination
30
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Chloroplast agglutination
Crude fresh leaf sap from diseased plant
Antiserum
Chloroplast fragments
clump together
31
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
The reactants antiserum and virus solution are placed in well cut
in the agar (containg 085 NaCl and 002 sodium azide) in
Petri plate
Antibody and virus diffuse into the agar from the adjacent wells
Where they meet precipitation zones in the form of white band
are formed
Gel diffusion test
Oservation
a) Bands Identical or closely related
b) Spurs Distantly Related
c) Intersect Unrelated 32
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Radial or single diffusion
Double or Ouchterlony diffusion33
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Immuno-electrophoresis
34
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
ELISA Enzyme- Linked Immuno-Sorbent Assay
ELISA was initially applied for plant viruses by MF Clark and
Adams (1976)
Sensitive detects at concentration of 1-10 ngml
It involves an enzyme-mediated colour change reaction to detect
antibody binding
Degree of colour change usually measured quantitatively in
spectrophotometer at 405 nm
Ward et al 200335
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
DAS ELISA Double antibody sandwich ELISA
Direct ELISA
First time describe by Clark and Adams in 1977
p-nitrophenyl
phosphatep-nitrophenol Ward et al 2003
36
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
DAC ELISA direct antigen coating ELISA
Indirect ELISA
Stand for Easy to rapid assay
Ward et al 200337
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Combination of electron microscopy
and serology
First time described by Derrick in1973
Virus and antiserum are reacted
together
Antigen are trapped onto grid coated
with specific antiserum negatively
stained (Uranyl acetate -1) and the
result viewed in the EM
Immuno Sorbernt electron microscope (ISEM)
Tubular particle of beet
necrotic yellow vein virus
38
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Dot Immunoblotting Assay (DIBA) OR Dot ELISA
Substrate Nitro tetrazolium
BCIP
39
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Lateral flow technique
bull The principles used for rapid lateral flow devices are primarily
those of ELISA
bull Various types of filters are used as the solid support for the
initial binding reaction
A lateral flow device test kit developed by Central Science
Laboratory UK permits detection of R solanacearum in a 3-
minute40
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
41
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Ouchterloniersquos double diffusion test
I ndash Healthy cane
extract
II ndash Control blood
serum of rabbit
III IV V ndash Antigen
of host pathogen
A ndash Antibody
raised against host
pathogen
Lingayya and Naik 2002
42
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Detection of Colletotrichum falcatum infection in sugarcane
tissue by DAC - ELISA
Lingayya and Naik 2002 43
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
TDA = 3 X standard deviation of
healthy sample + mean value for
healthy sample
44
FUNGUS
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Advantage of ELISA
It is sensitive
Semiautomatic technique
Application against large number of sample
Reproducible
Qualitative amp Quantitative
Suitable for automation high speed
No radiation hazards
45
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Immunofluorescence
The intercellular location amp
distribution of viruses
Globulins mixed with a
fluorescent dye (Fluorescein
isothiocyanate and Rhodamine
B)
Introduced into the infected
cellstissue with antigen and
antibody reaction fluorescence
takes place
46
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Flow cytometry
Cell suspensions are filtered to remove large particles then
stained with fluorochrome-labelled antibodies
Fluorescent markers for viability
Stains such as propidium and
hexidium iodide for red fluorescent
staining of dead cells
Carboxy fluorescein diacetate and calcein
AM for green fluorescent staining of
viable cells can be used to differentiate
live from dead cells
Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)
47
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
HYBRIDIZATION BASED METHODS
48
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
49
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Southern blotting
bull Professor Sir Edwin Southern
developed this method in 1975
bull This method Involves separation
transfer and hybridization
bull Detection of a specific DNA
sequence in DNA samples
bull Combines agarose gel
electrophoresis for size
separation of DNA and
hybridization with probeProfessor Sir Edwin Southern
50
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
51
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Northern Blotting
Northern blotting is a technique for detection of specific RNA
sequences
Northern blotting was developed by James Alwine and George
Stark at Stanford University (1979)
Electrophoresed RNA is blotted on membrane and hybridized
52
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
53
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Western blotting
bull Western blotting (1981) is an immunoblotting technique which
rely on the specificity of binding between a protein of interest
and a probe (antibody raised against that particular protein) to
allow detection of the protein of interest in a mixture of many
other similar molecules
bull The SDS (Sodium dodecyl sulphate) page technique is a
prerequisite for western blotting
54
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Steps in western blotting
Detected
through
auto-
radiography
55
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
DNA Microarray
56
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Microarrays for Rapid Identification of Plant
Viruses
Neil Boonham Jenny Tomlinsonand Rick Mumford
Central Science Laboratory Sand Hutton York YO41 1LZ
United Kingdom
57
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Boonham et al 2007
A schematic diagram detailing a simple approach to virus
detection using a microarray58
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
A microarray designed to detect and
discriminate a range of small spherical
viruses Eg Broad bean wilt virus 2
Indicator host Chenopodium quinoaA small spherical virus was identified
using electron microscopy
Boonham et al 200759
Positive control spots
Detection of virus
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Sensitivity comparison between ELISA and microarray
Boonham et al 2003
Dilution end
point
Histogram showing localbackground fluorescence for the11600 dilution of RNA
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
BIOCHEMICAL METHODS
bull FAME analysis
bull BIOLOG
bull Volatile compound
Biochemical techniques
61
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
What is a BIOLOG
First and only bacterial identification system to identify both gram positive
and gram negative bacteria with a single universal test kit
Add cells
96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the
positive well
62
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Fatty Acid Methyl Ester ( FAME ) analysis
Change in the fatty acid profile represent a change in the
microbial population
63
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Detection of Diseased Plants by Analysis of Volatile
Organic Compound Emission
RMC Jansen J Wildt IF Kappers
HJ Bouwmeester JW Hofstee
and EJ van Henten
64
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Emission of volatile organic compounds (VOCs)
from non-infected and Botrytis cinereandashinfected
tomato plants
Jansen et al 2011 65
Damaged cell membranes
Local emission of several lipoxygenase (LOX)
oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )
Characterize diseases due to release of VOCs
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Surface Plasmon Resonance (SPR)
The Surface Plasmon Resonance
(SPR) sensor is used for label free
detection and real-time monitoring
Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring
In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface
66Is it the specific protein the virus fragment or the virion itself
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Development of Surface Plasmon Resonance (SPR) Based Immuno-
Sensing System for Detection of Fungal Teliospores of Karnal Bunt
(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1
1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences
amp Humanities G B Pant University of Agriculture amp Technology Pantnagar
Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala
Dehradun Uttarakhand India
Journal of Biosensors amp
Bioelectronics
67
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Experiment conducted Interaction of teliosporic wall
antigen with the anti-teliosporic antibody immobilized on sensor
chip
The interaction of antigen at a concentration of 80 40 20 10 50 25
125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody
on sensor chip was examined
Observation
bull The responses increased in proportion to the concentration of teliosporic
antigen due to the change of the refractive index near the SPR sensor chip
68
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
SPR sensor response after the interaction of different concentrations of antigen over
the immobilized antibody (1 500)
Singh et al 2012
(a) 0312
ngμl
(b)125 ngμl
(c) 5 ngμl
(d) 20 ngμl
(e) 80 ngμl
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Advantages of SPR
Major advantages
bull Rapid real-time
bull Non-labeling analysis
bull Miniaturization for portable application
70
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
71
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Phytophthora and Pythium Test Kits 05 per cent of a
plant roots are infected
Tests for Phytophthora Pythium and Rhizoctonia root and crown decay
fungi can be performed on-site by growers in about 10 minutes
(A) Collect and grind samples using abrasive pads
(B) Fold pads and insert them into the extraction solution
(C) Apply solutions to detector
(D) Examine detector dots for color change72
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
Molecular methods for detection of plant
pathogensmdashWhat is the future
Strategies are needed on how to exploit deduced genomics and
proteomics supported by in silico analysis for establishing
rational disease control measures
The reliability of each specific on-the-spot diagnostic method
needs to be validated before results are used exclusively to
implement costly disease control strategies andor regulatory
actions
73
74
75
74
75
75