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

• Published in: Journal of virological methods Vol. 161; no. 2; pp. 289 - 296
• Main Authors: Mortimer-Jones, Sheila M., Jones, Michael G.K., Jones, Roger A.C., Thomson, Gordon, Dwyer, Geoffrey I.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01-11-2009; Elsevier
• Subjects: Biological and medical sciences; Carlavirus - genetics; Carlavirus - isolation & purification; DNA Probes; Fundamental and applied biological sciences. Psychology; Luteoviridae - genetics; Luteoviridae - isolation & purification; Lycopersicon esculentum; Lycopersicon esculentum - virology; Microbiology; Multiplex real-time RT-PCR; Plant Leaves - virology; Plant Roots - virology; Plant Viruses - genetics; Plant Viruses - isolation & purification; Potato leafroll virus; Potato leafroll virus (PLRV); Potato tubers; Potato virus S; Potato virus S (PVS); Potato virus X; Potato virus X (PVX); Potexvirus - genetics; Potexvirus - isolation & purification; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - genetics; RNA, Viral - isolation & purification; Sensitivity and Specificity; Solanum tuberosum; Solanum tuberosum - virology; Techniques used in virology; Tomato spotted wilt virus; Tomato spotted wilt virus (TSWV); Tospovirus - genetics; Tospovirus - isolation & purification; Virology; Multiplex real-time RT-PCR; Potato leafroll virus (PLRV); Potato tubers; Potato virus X (PVX); Potato virus S (PVS); Tomato spotted wilt virus (TSWV); Multiplex real-time RT―PCR; Plant pathogen; Microbiology; Tomato spotted wilt virus; Bunyaviridae; Flexiviridae; Dicotyledones; Angiospermae; Luteoviridae; Solanaceae; Reverse transcription polymerase chain reaction; Quantitative analysis; Multiplex polymerase chain reaction; Solanum tuberosum; Potexvirus; Tospovirus; Potato leafroll virus; Method; Real time; Polerovirus; Carlavirus; Virus; Potato virus X; Spermatophyta; Tuber plant; Detection; Potato virus S
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2009.06.027

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.