Optimization of a direct real-time quantitative reverse transcription polymerase reaction (DRT-qPCR) assay for the detection of grapevine rupestris stem-pitting associated viruses (GRSPaV) in grapevine

A single step Direct real-time quantitative reverse transcription quantitative polymerase chain reaction (DRT-qPCR) was applied and optimized for the detection of Grapevine rupestris stem-pitting associated virus (GRSPaV) in grapevine. A probe and primer set were designed from conserved genomic regions from eight GRSPaV accessions from GenBank and used with the Direct Plant Extraction Buffer (DiPEB) system in place of total nucleic acid extraction using commercial kits to identify 14 grapevine accessions and field grapevine isolates for the presence of GRSPaV. Optimal detection of GRSPaV was obtained using foliar tissue macerated first in ELISA extraction buffer that was further diluted in DiPEB buffer (1:9 v/v). Middle-aged grape leaves on young canes gave acceptably low Cq (quantification cycle) values, with no significant differences detected between bisected half leaves, refrigerated and frozen leaves, and frozen leaf macerates made in ELISA buffer. Cq values of macerates derived from cambial tissue were comparable to those of leaf tissue macerates. The DRT-qPCR was shown to be a reproducible, specific test capable of detection of as little as 4.59 pg of viral RNA. This is the first study to use this simplified method for the detection of viruses that infect grapevine. Because this system does not require the use of a commercial nucleic acid extraction kit, it provides savings in cost and time allowing higher throughput testing.