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.

中文翻译：

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等温逆转录-重组酶聚合酶扩增法快速，灵敏地检测马铃薯中的Y病毒。

在这项研究中，开发了等温逆转录重组酶聚合酶扩增（RT-RPA）测定法，用于在等温条件下有效，准确地检测马铃薯病毒Y（PVY）。这种RT-RPA分析比常规逆转录聚合酶链反应（RT-PCR）分析更有效，因为扩增反应可以在不到20分钟的时间内完成。而且，与需要热循环仪在特定温度阶段进行DNA扩增的PCR不同，RPA分析可以在25至40°C的等温条件下进行。在夏季，一个简单的仪器（例如加热块或水浴）或什至非仪器条件（例如人的手或房间内/室外的台式）都可以满足RPA的温度要求。此测定法的灵敏度与常规RT-PCR相当，并且可以从从PVY感染的马铃薯叶片组织中提取的至少2 pg总RNA中检测出该病毒。然后，通过酶联免疫吸附测定（ELISA）筛选，使用田间马铃薯叶和休眠断裂的新芽样品评估新开发的RT-RPA的功效。在164个PVY-ELISA阳性样品中，RT-RPA检测到157，而单纯RT-PCR检测到160，多重RT-PCR检测到154。在74个随机选择的PVY-ELISA阴性样品中，RT-RPA，单纯RT-PCR多重RT-PCR分别导致1、1和0阳性检测。总体而言，RT-RPA和两种RT-PCR测定法以及ELISA的一致性为96.6-98.7％，从而证明了RT-RPA对于大规模检测PVY的适用性，