Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan®MGB probes

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan®MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (103–1010 TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

► Development of real time RT-PCR for Tomato spotted wilt virus (TSWV).
► Estimation of the number of RNA copies of TSWV genomic segment M.
► Quantitation of TSWV in different host plants and the thrips vector.
► Quantitation of different TSWV genotypes in mixed infections.