Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato. (April 2020)

• Record Type: Journal Article
• Authors: Wang, Ying; Chen, Ruhao; Nie, Xianzhou; Zhong, Ziyang; Li, Chunyan; Li, Kun; Huang, Wei; Fu, Xingyu; Liu, Jun; Nie, Bihua
• Is Part Of: Molecular and cellular probes. Volume 50(2020)
• Publication Date: 2020-04
• Subjects: Potato -- Potato virus Y -- Reverse transcription-recombinase polymerase amplification (RT-RPA) -- Tuber sprout(s) -- Diagnosis; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2019.101505
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: 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. Highlights: An isothermal RT-RPA method has been developed for detection of PVY CP gene of all known strains. This method could amplify the PVY target in less than 20 min with excellent flexibility in terms of temperature requirement ranging from 25 to 40 ℃. This was the first report to evaluate the RT-RPA method for PVY detection at an under-equipped condition by using large scale of field samples. Except for the RPA reaction, other steps of the assay procedure still needed to be optimized and simplified for fast on-site detection of PVY.