Development of Polymerase Chain Reaction for Rapid Detection of Grapevine

Specific PCR primers for GLRaVs 1 through 5 were designed, tested and optimized. One step RT-PCR methodology was also developed and the reagents and testing conditions were optimized. Beginning last year we have started testing newly introduced and tissue cultured grape materials using this one step RT-PCR methodology for the detection of these viruses. The information also has been transferred to two different private laboratories in Northern California for their use in testing grower’s material, and another laboratory established by a nursery operation. In our experience we found that RT-PCR still gives false negative results. We found that sometimes the titer of virus in grapevine tissue is lower than detection threshold, especially on many different rootstocks. However, to improve the sensitivity of RE_{CR for the detection of GLRaVs, we developed nested RT-PCR methodology. We designed nested primers for these viruses and our preliminary test results indicated that this method was 10-100 folds more sensitive that regular RT-PCR. We are in the process of optimizing conditions for this method. In order to develop a reliable PCR detection method, it is very important to identify variations among different isolates (or strains) of each one of these GLRaVs and accordingly design PCR primers that could detect all diverse isolates. In the past year sequences from the coat protein gene from 15 different isolates of GLRaV-s were compared. It was found that 13 of the tested isolates were very similar in their coat protein sequences, ranging from 99.5%to 100%nucleotide sequence similarites among them. These 13 isolates had 89.2% similarities to the published GLRaV2 coat protein sequence. The remaining two isolates were more divergent and they had 89.4%abd 89.7% similarities to other 13 isolates and 99.5%and 92.5%similarities to the published GLRaV2.