Department of Life Sciences, University of Trieste, Trieste, Italy; Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
Institute for Sustainable Plant Protection, CNR, Bari, Italy.
J Virol Methods. 2018 Jul;257:42-47. doi: 10.1016/j.jviromet.2018.04.008. Epub 2018 Apr 11.
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.
检测欧盟植物材料认证中规定的四种葡萄病毒（GLRaV-1、GLRaV-3、GFLV 和 ArMV）需要敏感和特异的诊断工具。本研究基于 EvaGreen 结合染料，开发了一种在实时单管扩增中同时检测的策略。对扩增产物进行熔解曲线分析（MCA），可在多重分析中定性检测四种不同的病毒靶标。质粒稀释测定法计算分析灵敏度，其扩增阈值高达目标序列 100 个拷贝。一小部分田间葡萄样本，分别已知感染目标病毒混合物或未感染，成功地进行了检测，以评估扩增子的 Tm。
