Isothermal Diagnostics

Non-PCR based methods for

GM detection

Co-Extra International

Conference

2-5 June 2009 Paris

GM detection

Presented by Guy Kiddle

L u m o r a

Molecular made easy

The Convention?qPCR / fluorescence detection

Alternatives

Chemical Imaging

Near Infrared Spectroscopy (NIR; CRA-W)

Molecular Detection (Isothermal Nucleic Acid Amplifications iNAATs)


Conference

2-5 June 2009 Paris

NASBA Implemented microarray analysis (NAIMA; NIB)

Loop mediated amplification (LAMP; Lumora; NIAB)

Chimeric Displacement Amplification (RDC; Lumora)

Coupled to a bioluminescence reporter - BART

Polymerase Chain Reaction

Detection

- +


Conference

2-5 June 2009 Paris

- +

Chemical Imaging


Conference

2-5 June 2009 Paris

Chemical Imaging

(NIR)

Near Infrared Spectroscopy (NIR)

Instrumentation


Conference

2-5 June 2009 Paris

ImagingPrincipal Component AnalysisSpectral Data

Sa

mp

les

Non GM

Transgenic

Non-GM

HHIA (2)4+HHIA (2)1

HQ2A1

• No need for prior knowledge

• Simple operation

• Non invasive

Advantages of NIR


Conference

2-5 June 2009 Paris

TransgenicNon-GM

Non GM

Transgenic

HCIB (6)1+ HCIB (5)1

• High throughput

• Identifies GM seed (80% effective)

• Quantifiable

Isothermal Amplification

& Detection

L u m o r a

Molecular made easy


Conference

2-5 June 2009 Paris

& Detection

NASBA

NASBA implemented microarray

analysis (NAIMA)

GM DNA tagged RNA promoter (PCR)

T7 RNA polymerase


Conference

2-5 June 2009 Paris

NASBA

Real-time NAIMA

SYBR reporter


Conference

2-5 June 2009 Paris

Melt Curve

•Rapid amplification (25min)

•Real-time analysis

• Sensitive

Advantages of NAIMA


Conference

2-5 June 2009 Paris

• Sensitive

• Robust chemistry (processed / raw / trace / carrier)

• Multiplex platform

Loop Mediated Isothermal Amplification

104 103 102 10 1 0105


Conference

2-5 June 2009 Paris

Concatamers ++++++ PPi

M

20

5

29

7

62

2

M 20

5

29

7

62

2

ve

Engineered NOS-p targets Resolved LAMP amplicon

Effect of DNA stem length (LAMP)


Conference

2-5 June 2009 Paris

600

300

200

M 20

5

29

7

62

2

-ve

25

200

100

400

600800

1000 R1000

R25

Bioluminescent Assay in Real-Time (BART)


Conference

2-5 June 2009 Paris

Bespoke Instrument / Software


Conference

2-5 June 2009 Paris

GM-LAMP-BART (Sensitive / Quantifiable)


Conference

2-5 June 2009 Paris

GM-LAMP-BART (Robust)

45.0

55.0

65.0

75.0

85.0

Pe

ak

time

(min

)

25.0

30.0

35.0

Cy

cle

tim

e (

min

)

qPCR (ADH1) LAMP-BART (ADH1)

SDS CTAB NaCl Xylan Starch Humic

Acid

CaCl2 SDS CTAB NaCl Xylan Starch Humic

Acid

CaCl2


Conference

2-5 June 2009 Paris

iNAATs are more tolerant to classical PCR inhibitors

Inhibitor

15.0

25.0

35.0

15.0

20.0

Nd Nd Nd Nd Nd Nd Nd Nd Nd

GM-LAMP-BART (Portable)


Conference

2-5 June 2009 Paris

PDQ

0

10

20

30

40

50

60

70

80

90

100

10 100 1000 10000

qPCRphotodiode

• Rapidly amplifies target GM elements

• Offers real-time alternative for GM quantification

• Simple instrumentation

Advantages of GM-LAMP-BART


Conference

2-5 June 2009 Paris

• Simple instrumentation

• Robust chemistry

• Field-able

GM-RDC-BART (Development)

SNP detection


Conference

2-5 June 2009 Paris

LAMP-BARTAll of my colleagues at Lumora,

Jim Murray

Manuella Rizolla

Cathy Moore

and David Lee (Cambridge University / Lumora Ltd / National Institute of Agricultural Botany)

Acknowledgements

NAIMADany Morissette


Conference

2-5 June 2009 Paris

Dany Morissette

and Kristina Grüden (National Institute of Biology, Slovenia)

NIRGilbert Berben

(Agricultural Research Centre of Gembloux, Belgium)

All of the partners above would like to express our gratitude to

the organizing committee and to the EU Commission for funding

the presented work.

Isothermal Diagnostics

Documents

Transcript of Isothermal Diagnostics