Development of Methodologies for Rapid Detection

The efficiency of ELISA for detecting grapevine leafroll associated viruses was compared with bioassay index on field indicator Cabernet Franc. The test results indicated a good correlation between ELISA and field index. To overcome the shortage of polyclonal antibodies for grapevine leafroll associated viruses (GLRaV) types (T) Til, Till and TIV, viruses have been isolated and purified and Rabbits have been immunized. The preliminary evaluation of these antisera indicated that the antiserum to Till has a relatively high titer, to TIV has low titer and no specific titer was found for Til. A polymerase chain reaction (PCR) technique which is a very sensitive and reliable method for the detection of plant viruses has been developed for the detection of grapevine fanleaf virus (GFLV) in grapevine tissue. This technique was able to detect 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) for the detection of GFLV, GLRaV Till and grapevine virus A (GVA). In this technique the lengthy process of nucleic acid extraction required for PCR has been eliminated. The results showed that this modification works quite well for the detection of these 3 viruses. Attempts are underway to develop PCR for the detection of GLRaV Til and TIV.