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

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 develo...

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Veröffentlicht in:Journal of virological methods 2007-06, Vol.142 (1), p.1-9
Hauptverfasser: Agindotan, Bright O., Shiel, Patrick J., Berger, Philip H.
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Shiel, Patrick J.
Berger, Philip H.
description 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.
doi_str_mv 10.1016/j.jviromet.2006.12.012
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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
title Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan ® real-time RT-PCR
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