A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously

Abstract

A high throughput, real-time multiplex, single tube RT-PCR assay was developed for simultaneous detection of Potato leafroll virus (PLRV), Potato virus X (PVX) and Potato virus S (PVS) in potato leaves and tubers, and Tomato spotted wilt virus (TSWV) in potato tubers and tomato leaves. The test uses four different fluorescently labelled TaqMan® probes. Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 × 101 to 8 × 109 copies of PVX and PVS, 1 × 102 to 1 × 1010 copies of PLRV and 1 × 103 to 1 × 1010 copies of TSWV). For each individual assay, the inter-assay reproducibility was high, with a coefficient of variation of the combined assays of <2%. Total RNA was extracted rapidly and efficiently from bulked samples equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV simultaneously in a single assay. The multiplexed assay was validated in blind studies with leaves and tubers. This high-throughput test is accurate and sensitive, and provides seed potato industries with a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers. © 2009 Elsevier B.V.