Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis

作者：Aloisio M; Morelli M; Elicio V; Saldarelli P; Ura B; Bortot B; Severini G M; Minafra A^*

来源：Journal of Virological Methods, 2018, 257: 42-47.

DOI：10.1016/j.jviromet.2018.04.008

• Multiplex Real-time PCR
• Melting curve
• EvaGreen
• Grapevine
• Viruses

The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.

• 出版日期2018-7