Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato.

Autor: Wang Y; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Chen R; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Nie X; Fredericton Research and Development Center, Agriculture and Agri-Food Canada, 850 Lincoln Road, P. O. Box 20280, Fredericton, New Brunswick, E3B4Z7, Canada., Zhong Z; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Li C; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Li K; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Huang W; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Fu X; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Liu J; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China., Nie B; National Center for Vegetable Improvement (Central China), Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, China. Electronic address: nbihua@mail.hzau.edu.cn.
Jazyk: angličtina
Zdroj: Molecular and cellular probes [Mol Cell Probes] 2020 Apr; Vol. 50, pp. 101505. Date of Electronic Publication: 2020 Jan 02.
DOI: 10.1016/j.mcp.2019.101505
Abstrakt: In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6-98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.
(Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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