Detection of grapevine closterovirus A in infected grapevine tissue by reverse transcription-polymerase chain reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was successfully applied to detection of GVA RNA in nucleic acid extracts of infected grapevines. In particular, an artificially synthesized DNA primer set designed to amplify a GVA cDNA fragment of 430 base pairs, specifically detected GVA RNA sequences in extracts from infected grapevine tissues such as leaves from in vitro-grown explants, leaves from greenhouse-grown rooted cuttings, and bark scrapings of mature canes from field-grown vines. The detection limit of GVA RNA by RT-PCR was estimated to be 200 fold higher than that obtained by molecular hybridization or ELISA.