Wang Yahui, Wang Qing, Bergmann Sven M, Li Yingying, Li Bo, Lv Yuefeng, Yin Jiyuan, Yang Guang, Qv Yang, Wang Yingying, Zeng Weiwei
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development of Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China; College of Fisheries, Tianjin Agriculture University, Tianjin, China.
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development of Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Mol Cell Probes. 2021 Dec;60:101776. doi: 10.1016/j.mcp.2021.101776. Epub 2021 Nov 3.
Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Early and accurate diagnosis is an important priority for TiLV disease control. In order to evaluate the methodology in the molecular diagnosis of TiLV, we compared newly developed quantitative real-time PCR (qPCR) and real-time recombinase polymerase amplification (real-time RPA) assays regarding their sensitivities, specificities and detection effect on clinical samples. Real-time RPA amplified the target pathogen in less than 30 min at 39 °C with a detection limit of 620 copies, while qPCR required about 60 min with a detection limit of 62 copies. Both assays were specific for TiLV and there were no cross-reactions observed with other common fish pathogens. The assays were validated using 35 tissue samples from clinically infected and 60 from artificially infected animals. The sensitivities for the real-time RPA and qPCR assays were 93.33 and 100%, respectively, and the specificity was 100% for both. Both methods have their advantages and can play their roles in different situations. The qPCR is more suitable for quantitative analysis and accurate detection of TiLV in a diagnostic laboratory, whereas real-time RPA is more suitable for the diagnosis of clinical diseases and preliminary screening for TiLV infection in poorly equipped laboratories as well as in fish farms.
罗非鱼湖病毒（TiLV）是一种新出现的病原体，在全球罗非鱼养殖业中导致高死亡率和经济损失。早期准确诊断是控制TiLV疾病的重要优先事项。为了评估TiLV分子诊断方法，我们比较了新开发的定量实时PCR（qPCR）和实时重组酶聚合酶扩增（实时RPA）检测方法在灵敏度、特异性以及对临床样本的检测效果方面的差异。实时RPA在39℃下不到30分钟即可扩增出目标病原体，检测限为620拷贝，而qPCR则需要约60分钟，检测限为62拷贝。两种检测方法对TiLV均具有特异性，未观察到与其他常见鱼类病原体的交叉反应。使用来自临床感染动物的35份组织样本和来自人工感染动物的60份组织样本对检测方法进行了验证。实时RPA和qPCR检测方法的灵敏度分别为93.33%和100%，特异性均为100%。两种方法都有各自的优点，可在不同情况下发挥作用。qPCR更适合在诊断实验室中对TiLV进行定量分析和准确检测，而实时RPA更适合在设备简陋的实验室以及养鱼场中对临床疾病进行诊断和对TiLV感染进行初步筛查。
