A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously
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...
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Published in | Journal of virological methods Vol. 161; no. 2; pp. 289 - 296 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Kidlington
Elsevier B.V
01.11.2009
Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | 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
×
10
1 to 8
×
10
9 copies of PVX and PVS, 1
×
10
2 to 1
×
10
10 copies of PLRV and 1
×
10
3 to 1
×
10
10 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0166-0934 1879-0984 |
DOI: | 10.1016/j.jviromet.2009.06.027 |