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

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...

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Bibliographic Details
Published inJournal of virological methods Vol. 257; pp. 42 - 47
Main Authors Aloisio, M., Morelli, M., Elicio, V., Saldarelli, P., Ura, B., Bortot, B., Severini, G.M., Minafra, A.
Format Journal Article
LanguageEnglish
Published Netherlands Elsevier B.V 01.07.2018
Subjects
EvaGreen
Grapevine
Melting curve
Multiplex Real-time PCR
Viruses
Multiplex Real-time PCR
Viruses
Melting curve
EvaGreen
Grapevine
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Summary: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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ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2018.04.008