Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan ® real-time RT-PCR

• Published in: Journal of virological methods Vol. 142; no. 1; pp. 1 - 9
• Main Authors: Agindotan, Bright O., Shiel, Patrick J., Berger, Philip H.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.06.2007; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; DNA Primers; Fundamental and applied biological sciences. Psychology; Microbiology; Multiple virus detection; Multiplex detection; Plant Diseases - virology; Plant Viruses - classification; Plant Viruses - genetics; Plant Viruses - isolation & purification; Potato tubers; Potexvirus - genetics; Potexvirus - isolation & purification; Potyvirus - genetics; Potyvirus - isolation & purification; Real-time RT-PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA Viruses - classification; RNA Viruses - genetics; RNA Viruses - isolation & purification; RNA, Viral - analysis; RNA, Viral - isolation & purification; Sensitivity and Specificity; Solanum tuberosum - virology; Taq Polymerase; TaqMan; Techniques used in virology; Virology; Real-time RT-PCR; Multiple virus detection; Potato tubers; TaqMan; Multiplex detection; Plant pathogen; Microbiology; Dicotyledones; Angiospermae; Luteoviridae; Potyvirus; Solanaceae; Potato leaf roll virus; Reverse transcription polymerase chain reaction; Solanum tuberosum; Method; Real time; Polerovirus; Virology; Virus; Potato virus Y; Spermatophyta; Tuber plant; Detection; Potyviridae
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2006.12.012

The requirements of sprouting dormant potato tubers for biological or serological assays or RNA extraction for nucleic acid and PCR assays add to the cost of virus screening. Recently, cheaper, reliable and more rapid methods for the screening of potato tuber-seed pieces for viruses have been developed that do not require sprouted tubers for indexing, including TaqMan ® real-time RT-PCR. Although the assays are often designed for minimal time and reagent use, they still require a time-consuming and laborious RNA extraction step. This paper describes an assay where four common potato-infecting viruses, Potato leafroll virus, Potato virus A, Potato virus X and Potato virus Y, were detected simultaneously from total RNA and saps of dormant potato tubers in a quadruplex real-time RT-PCR. Factors critical for the detection of these viruses in saps of dormant potato tubers included: optimum dilution and inhibition of RNAses, and the optimization of the reverse transcription and PCR steps. Potato virus detection directly from tuber saps was comparable to that from purified total plant RNA, and this represents significant savings of time and expense. The TaqMan ® system developed in this study detected between 200 and 400 copies of potato virus RNA.