Development and validation of a high throughput, one-step, quantitative real-time RT-PCR assay for the simultaneous detection of PLRV, PVX, PVS and TSWV with a rapid RNA extraction method directly from bulked potato tuber samples
Mortimer-Jones, S.M., Jones, M.G.K., Jones, R.A.C. and Dwyer, G. (2008) Development and validation of a high throughput, one-step, quantitative real-time RT-PCR assay for the simultaneous detection of PLRV, PVX, PVS and TSWV with a rapid RNA extraction method directly from bulked potato tuber samples. In: 8th Plant Virology Workshop, 19 - 22 November 2008, Rotorua, New Zealand.
Potato is important in Western Australia both for domestic food production and export. Four viruses diminish tuber yield locally, Potato leaf roll virus (PLRV), Potato virus X (PVX), Potato virus S (PVS) and Tomato spotted wilt virus (TSWV). A real-time multiplex, single tube RT-PCR assay for the detection of these viruses from potato leaves and tubers was developed using Cy5-, FAM-, JOE- and ROX-labelled TaqMan probes. The copy numbers for transcripts were quantified with a dynamic range of 8x101 to 8x109 copies of PVX and PVS, 1x102 to 1x1010 copies of PLRV and 1x103 to 1x1010 copies of TSWV. The inter-assay reproducibility was high, with a coefficient of variation (CV) of <2%. Total RNA was rapidly and efficiently extracted from bulked tuber samples for the reliable detection of one or more of the viruses. These data indicate that this high-throughput test is accurate and sensitive, and will provide a cost-effective diagnostic tool for the seed potato industry.
|Publication Type:||Conference Item|
|Murdoch Affiliation:||Western Australian State Agricultural Biotechnology Centre|
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