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

Publication date: July 2018
Source:Journal of Virological Methods, Volume 257
Author(s): M. Aloisio, M. Morelli, V. Elicio, P. Saldarelli, B. Ura, B. Bortot, G.M. Severini, A. Minafra
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.