Development of Polymerase Chain Reaction for Rapid Detection of Grapevine
Polymerase chain reaction (PCR) is used for the detection of plant viruses. It is a very sensitive, reliable, and universal technique compared to ELISA and other available plant virus detection methods. PCR can be designed to detect more than one type of virus in a single reaction using universal primers (this criteria is lacking in ELISA). In the past year we have developed PCR for the detection of grapevine leafroll associated virus (GLRaV) types 1, 2, 3, 4, and 5. We also have designed universal reactions that could detect GLRaV 1+2 and 4+5 each in a single reaction. We are currently in the process of identifying various strains of each type of GLRaV. By identifying these strains for each virus type, we can improve the reliability and reproducibility of PCR technique. We have also developed a simple method of sample preparation from grapevine tissue for PCR detection of grapevine viruses. This simplified sample preparation method is no more complex or labor intensive than preparing grapevine samples for ELISA. We also have further simplified PCR reaction and developed a simple one step reaction procedure. In this method the chance of cross contamination between samples is reduced significantly and test results are available in under four hours. We have started using PCR methodology for the detection of different viruses in newly introduced grapevine materials to FPMS. We also use the same technique to test all the plants generated from tissue culture for virus elimination.