Detection of Several Important Viral Agents by Real-time PCR in Aegean Vineyards

Abstract

In this study, Grapevine fanleaf nepovirus (GFLV), Grapevine leafroll associated-virus 1 (GLRaV-1), Grapevine leafroll associated-virus 2 (GLRaV-2) and Grapevine leafroll associated-virus 3 (GLRaV-3) which were the most important viral diseases in Aegean viticulture production areas were determined with Syber Green I based Real-time PCR methods in a rapid and reliable way. For this purpose, separately GFLV, GLRaV-1, GLRaV-2 and GLRaV-3 infected grapevine samples were used which were previously detected by DAS-ELISA and conventional RT-PCR. Healthy plant sample was also used for negative control. The results of Syber Green I based Real-time PCR were confirmed by melting curve analyses which were performed to each samples right after Real-time PCR protocol. Result of melting curve analyses, non specific amplifications such as primer dimers were determined and false positive results were ignored. Detection of all above virus agents were successfully performed after optimisation assays with Syber Green I based Real-time PCR.