Optimization of a real-time RT-PCR assay and its comparison with ELISA, conventional RT-PCR and the grow-out test for large scale diagnosis of Potato virus Y in dormant potato tubers.
Singh, M., Singh, R.P., Fageria, M.S., Nie, X., Coffin, R.H., and Hawkins, G. (2013). "Optimization of a real-time RT-PCR assay and its comparison with ELISA, conventional RT-PCR and the grow-out test for large scale diagnosis of Potato virus Y in dormant potato tubers.", American Journal of Potato Research, 90(1), pp. 43-50. doi : 10.1007/s12230-012-9274-z Access to full text
A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVYO, PVYN, PVYN:O (= PVYN-Wi) and PVYNTN. Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25.75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, growout testing takes approximately 6-8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, realtime RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory.
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