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.
Department of Plant Pathology, University of California, Davis, CA 95616, USA.
Fatima
F
Osman
Adib
A
Rowhani
D001483:Base Sequence; D017124:Conserved Sequence; D052677:Flexiviridae / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014758:Viral Core Proteins / Q000235:genetics; D014777:Virus Diseases / Q000821:virology*; D027843:Vitis / Q000821:virology*
D015345:Oligonucleotide Probes; D014758:Viral Core Proteins