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.
Osman
Fatima
F
Department of Plant Pathology, University of California, Davis, CA 95616, USA.
Rowhani
Adib
A
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