Development of multiplex real-time PCR for simultaneous detection of three Potyviruses in tobacco plants

Abstract

Aims: To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and quantification of Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV).

Methods and results: Specific primer and probe combinations for TEV and TVBMV were developed from the coat protein region of the viral genome. To detect PVY, a primer and probe combination PVY-Univ F, PVY-Univ R and PVY-Univ P for amplifying the coat protein region of the virus genome was employed. The detection limit of multiplex real-time PCR for these viruses was 10 copies μl(-1) of the standard plasmid. The multiplex reaction was successful in the detection of these three pathogens, with no non-specific amplification and cross-reaction.

Conclusions: This multiplex real-time PCR provides a rapid, effective, specific and sensitive method for the simultaneous detection and quantification of the three pathogens on infected tobacco plants.

Significance and impact of the study: This multiplex real-time PCR will be useful not only for diagnostic, ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, in particular during mix infection.