Regional Disease Surveillance workshop for banana Rwanda 25 th -29 th January IITA-pathology,Uganda

Transcription

1 Regional Disease Surveillance workshop for banana Rwanda 25 th -29 th January 2010 Fen Beed Idd Ramathani IITA-pathology,Uganda

2 Why lab-based diagnostic tools??? Field Diagnostic tools symptoms expression-bbtd BBTD infected banana plants Nitrogen deficient banana plants

3 Signs pathogen presences Bacterial ooze- Xanthomonas wilt of banana Xanthomonas campestris pv musacearum (Normal yellow and Orange colored Klebsiella bacteria Xanthomonas axonopodis pv. citri

4 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.

5 Basic characteristic of BBW Growth 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].

6 Microscopy Electron microscope Light/fluorescence microscopy with micromanipulation facilities Xanthomonas campestris pv musacearum Banana bunch top virus. ssdna viruses Nanoviridae Babuvirus

7 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.

8 ELISA methods for detection of BBTD Two 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. Ig attached to virus antigen is detected by the addition of enzyme-ig conjugates. 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 for quantitative estimations, due to the use of crude extract.

9 DAS-ELISA FOR DETECTION OF BBTD-VIRUS ELISA serological test for BBTD and BBW

10 ELISA plate

11 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

12 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.

13 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 BBTV Using Multiplex PCR with primer BBTV 1 primer,bbtv 2 primer 2 & Brep F,Brep R.

14 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 RNA 2. RT enzyme transcribes the RNA sequence to a complementary DNA sequence 3. These DNA copies are then used as the template for PCR.

15 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 ( 32 P), or nonradioactive label

16 DNA probe Technology is based On DNA:DNA hybridization, which involves the selective pairing of nucleotides between two ssdna to Form a dsdna molecule

17 Thank you Merci