Real-time RT-PCR (TaqMan®) assays for the detection of viruses associated with Rugose wood complex of grapevine

Real-time TaqMan® RT-PCR (TaqMan® RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus in the genus Foveavirus, Grapevine virus A, Grapevine virus B and Grapevine virus D in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan® RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan® RT-PCR for the detection of these viruses using four-fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan® RT-PCR was more sensitive and could detect viruses at 32- and 256-fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR. The use of an internal control (18S rRNA) allowed comparison of sample preparation protocols and amplification efficiencies between samples.