Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fishery Sciences, Nanning, 530021, China.
Technology Center of Yantai Customs, Yantai, 264000, China.
J Virol Methods. 2022 Feb;300:114362. doi: 10.1016/j.jviromet.2021.114362. Epub 2021 Nov 18.
A recombinase polymerase amplification (RPA) assay was established for the rapid detection of Decapod iridescent virus 1 using primers targeted to the virus's ATPase gene (ORF114R). Optimization experiments showed that the optimal amplification temperature of the RPA assay was 37 °C and that the reaction could be completed within only 15 min. The target band of 15 min. is bright enough. In order to shorten the operational reaction time, consequently, 15 min was the optimal amplification time for our new RPA assay for DIV1. Specificity tests showed that the RPA assay did not exhibit any cross-reactivity with other shrimp pathogens(TSV, MrNV, YHV-1, WSSV, EHP, AHPND, EHNV, RSIV, RGV and IHHNV). Sensitivity tests further showed that the detection limit of the new RPA assay was 200 copies/50 μL, indicating that this assay was more sensitive than a nested polymerase chain reaction (PCR) method. A total of 509 clinical samples were assayed using the RPA and the PCR assays; analysis showed that the RPA method could detect weak-positive samples more effectively than the PCR method. Collectively, these findings indicated that the RPA assay was fast, simple, specific, sensitive and has significant potentials for clinical and on-site testing.
建立了一种基于引物靶向病毒 ATPase 基因（ORF114R）的重组酶聚合酶扩增（RPA）检测方法，用于快速检测十足目虹彩病毒 1（Decapod iridescent virus 1，DIV1）。优化实验表明，RPA 检测的最佳扩增温度为 37°C，反应可在 15 min 内完成。目标条带在 15 min 时已经足够明亮。为了缩短操作反应时间，因此，我们的新 DIV1 RPA 检测的最佳扩增时间为 15 min。特异性试验表明，RPA 检测与其他虾类病原体（TSV、MrNV、YHV-1、WSSV、EHP、AHPND、EHNV、RSIV、RGV 和 IHHNV）没有任何交叉反应。敏感性试验进一步表明，新 RPA 检测的检测限为 200 拷贝/50 μL，表明该检测比巢式聚合酶链反应（PCR）方法更灵敏。使用 RPA 和 PCR 检测方法共检测了 509 份临床样本；分析表明，与 PCR 方法相比，RPA 方法能够更有效地检测弱阳性样本。总之，这些发现表明 RPA 检测方法快速、简单、特异性强、灵敏度高，具有重要的临床和现场检测潜力。
