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

J Virol Methods. 2008 Dec;154(1-2):69-75. doi: 10.1016/j.jviromet.2008.09.005. Epub 2008 Oct 30.


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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Conserved Sequence
  • Flexiviridae / genetics
  • Flexiviridae / isolation & purification*
  • Molecular Sequence Data
  • Oligonucleotide Probes / genetics
  • Plant Diseases / virology*
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Sensitivity and Specificity
  • Sequence Alignment
  • Viral Core Proteins / genetics
  • Virus Diseases / virology*
  • Vitis / virology*


  • Oligonucleotide Probes
  • Viral Core Proteins