Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

中文翻译：

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开发用于实时检测鲤鱼病毒春季病毒血症的实时逆转录重组酶聚合酶扩增法。

鲤鱼春季病毒血症（SVCV）是一种重要的病原体，可导致多种鱼类大规模爆发鲤鱼春季病毒血症（SVC），给水产养殖业带来巨大的经济损失。一种简单方便的检测方法对于SVCV诊断是必不可少的。在这项研究中，实时逆转录重组酶聚合酶扩增（RT-RPA）分析方法得到了开发和验证。设计了针对M基因保守区的引物和探针，并将其应用于在39°C下进行20分钟的实时RT-RPA分析。特异性分析表明未发现与鱼类其他致病病毒的交叉反应，表明该测定具有适当的特异性。体外转录的RNA标准品用于评估测定的灵敏度，检出限为102份/反应。为了进一步评估该分析，使用实时RT-RPA分析和实时RT-PCR方法测试了65个临床样品。观察到相同的检测结果，表明实时RT-RPA分析在临床样品检测中的潜在应用。这是有关用于SVCV检测的RPA检测的第一份报告，这种新开发的检测方法可在实验室和SVCV诊断领域中使用。