Regional Disease Surveillance workshop for bananaRwanda 25th -29th January 2010

Idd RamathaniIITA-pathology ,Uganda

Fen Beed

Why lab-based diagnostic tools??? Field Diagnostic tools – symptoms expression-BBTD

BBTD infected banana plants Nitrogen deficient banana plants

Signs –pathogen presences Bacterial ooze- Xanthomonas wilt of banana

Klebsiella bacteria Xanthomonas axonopodis pv. citri

Xanthomonas campestris pv musacearum(Normal yellow and Orange colored

Lab –based diagnostic methods (1) direct detection, Electron Microscopy morphology / immune electron

microscopy Light microscopy histological appearance - e.g.

inclusion bodies Antigen detection immunofluorescence, ELISA

Competitive methods Sandwich methods Antibody capture methods

Biochemical analysis (2) Molecular techniques for direct detection of pathogen

genomes.

Basic characteristic of BBWGrowth is inhibited by 0.1% triphenyl tetrazolium chloride.Metabolic characteristics include: catalase positive; oxidase negative; do not denitrify or reduce nitrate; produce small amounts of acid from various carbohydrates and

other carbon sources, but not from rhamnose, adonitol, sorbitol, dulcitol, meso-inositol, inulin or salicin.

Gluconate is not immediately metabolised and asparagine is not used as a sole source of carbon and nitrogen simultaneously. DNA G + C content

described at genus level ranges from 62.6 to 69.4% Contains fatty acids 11:0 ISO, 11:0 ISO 3OH and 13:0 ISO 3OH

that are unique to the Xanthomonas genus Bacteria were identified as Xanthomonas campestris(Xcm) by

fatty acid analysis [ID probability score approx. 0.9].

Microscopy

Light/fluorescence microscopy with micromanipulation facilities

Electron microscope

Xanthomonascampestris pvmusacearum

Banana bunch top virus.

› ssDNA viruses› Nanoviridae

› Babuvirus

Diagnosis of BBTD Using ELISA• ELISA is a test based upon an

antibody reaction to the BBTV virus particles in the banana plant sap (ELISA = Enzyme-linked Immunosorbent Assay)

• The ELISA test is a color change reaction to banana plant sap. Obtain from sections of suspect banana leaves as shown at right. Yellow indicates a positive reaction and that BBTV is present in the plant sap.

ELISA methods for detection of BBTDTwo forms of ELISA can be used Double Antibody Sandwich - DAS ELISA) form of direct ELISA - The wells are coated with antibody (or immunoglobulins = Ig) prepared

from antisera. The test sample is then added to allow trapping of virus antigen by the coated antibody in the well. This is followed by the addition of enzyme labelled antivirus Ig, which will attach to the trapped antigen.

- A substrate for the enzyme is now added for producing colour reaction. Peroxidase (00 450) or alkaline phosphatase (00 405) is used, which allow quantitative measurement through a spectrophotometric device (ELISA reader with. automated. photometer is often used.

Direct Antigen Coating DAC ELISA - In this direct antigen coating (OAC) technique, plant extracts prepared in a carbonate buffer are applied directly to the wells.

-In the second step, diluted unfractionated antiserum is added. Igattached to virus antigen is detected by the addition of enzyme-Igconjugates. This is the simplest procedure and can be completed within three hours. With 96 wells in a plate, as many as 16,000 samples could be tested in a day. This procedure, however, is not suitable forquantitative estimations, due to the use of crude extract.

DAS-ELISA FOR DETECTION OF BBTD-VIRUS

ELISA –serological test for BBTD and BBW

ELISA plate

Nucleic Acid-Based Methods

Polymerase chain reaction (PCR) (DNA viruses, fungi, bacteria)

Reverse transcriptase PCR (RT-PCR) (RNA viruses and viroids)

Hybridization: DNA probes RNA probes

Molecular Diagnosis of XCM & BBTV(PCR). It’s a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.

As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Molecular Diagnosis for XCM and BBTV•Molecular diagnosis of XCM•using rep-PCR with primers ERIC and BOX, ERIC 1R and ERIC 2 with sequences of 5'- ATGTTAAGCTCCTGGGGATTCAC-3' and 5'- AAGTAAGTGACTGGGGTGAGCG-3' respectively (Veracruz et al., 1996 and Restepo, 2000) Molecular diagnosis of BBTVUsing Multiplex PCR with primer BBTV 1 –primer ,BBTV 2 –primer 2 & Brep F ,Brep R.

Reverse-Transcriptase PCR (RT-PCR)• The nucleic acid is RNA, not DNA• Taq polymerase can only amplify DNA fragments• Reverse Transcriptase (RT) is used to make DNA copy of viral

RNA prior to PCR 1. DNA primer annealed to the viral RNA2. RT enzyme transcribes the RNA sequence to a complementary

DNA sequence3. These DNA copies are then used as the template for PCR.

Nucleic Acid Sequence Hybridization Powerful technique with widespread application Based on hybridization (binding) of complementary

DNA sequences

Requires probes, usually DNA fragment, which has sequence similarity only to DNA of the test organism or closely related organisms

DNA probe is labeled with radioactive phosphorous (32P), or nonradioactive label

DNA probeTechnology is basedOn DNA:DNAhybridization,which involves theselective pairing ofnucleotides betweentwo ssDNA toForm a dsDNAmolecule

Thank youMerci