Development of Methodologies for Rapid Detection of Grapevine Viruses
Additional experiment was completed to compare the efficiency of ELISA for detecting grapevine leafroll associated viruses with bioassay index on field indicator Cabernet Franc. The test results revealed a good correlation between ELISA and field index. We refined the grapevine leafroll associated viruses (GLRaV) purification methods so that we consistently obtained a cleaner, more concentrated virus preparation. We used this method to purify enough GLRaV types II and IV virus to immunize rabbits for the production of virus-specific antiserum. Our preliminary evaluation of the antisera indicated that both had very good virus-specific titer in ELISA. We were unable to obtain virus-specific cDNA clones for GLRaV types II and IV using dsRNA preparations. However, we have been successful in using purified virus preparations to produce cDNA clones for these two viruses. The clones have been screened and among numerous clones, we obtained one GLRaV type II clone believed to have sequence similar to part of the viral genome. We have made primers for polymerase chain reaction (PCR) from this clone and we are in the process of evaluating them. PCR, which is very sensitive and reliable method for the detection of plant viruses, has been developed for the detection of grapevine fanleaf virus (GFLV), GLRaV type III, grapevine virus A (GVA), and grapevine virus B (GVB) in grapevine tissue. This technique detects GFLV in a sample when one infected grapevine leaf was mixed with 200 healthy ones. We have modified and simplified the PCR methodology by combining immunology and PCR (immunocapture-PCR, IC-PCR). We have even further simplified the technique by labeling the PCR products with an enzyme enabling us to analyze the PCR products by an ELISA reader (colorimetric PCR). Our preliminary data showed that this modification increased the sensitivity and efficiency of the technique. In these modifications (IC-PCR and colorimetric PCR), the lengthy process of nucleic acid extraction required for PCR and analysis of PCR products by gel electrophoresis are eliminated. The results showed that this modification works quite well for the detection of these 4 viruses. In addition, combining the two methods, (IC-PCR and the colorimetric PCR) increases the sensitivity of detecting a virus-infected sample. In replicate experiments, we compared detection of IC-PCR products using colorimetric PCR versus gel electrophoresis. For all four viruses described, the colorimetric PCR was thousand¬fold more sensitive. In addition, problems with non-specific products and high background are eliminated with the use of colorimetric PCR, Attempts are underway to develop these PCR methods for the detection of GLRaV TII and TIV.