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Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis
M Aloisio1, M Morelli2, V Elicio3
1Department of Life Sciences, University of Trieste, Trieste, Italy; Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
Abstract:
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.
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