Tamer Cuneyt, Benkaroun Jessica, Kurucay Hanne Nur, Albayrak Harun, Weidmann Manfred
Department of Virology, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey.
Center for Virus Research, Glasgow, Scotland.
J Fish Dis. 2022 Aug;45(8):1065-1071. doi: 10.1111/jfd.13629. Epub 2022 Apr 25.
Viral diseases of fish cause significant economic losses in the aquaculture industry. Viral haemorrhagic septicemia virus (VHSV) is one of the most important viral diseases that affects more than 80 fish species. Detection of the disease, especially in the field, is critical to managing disease prevention and control programmes. Recombinase polymerase amplification (RPA) is an isothermal method with a very short amplification period and a single incubation temperature ranging from 37 to 42°C, which is a good alternative to the polymerase chain reaction (PCR). This study aimed to develop an RPA assay as sensitive as a real-time RT-PCR to detect VHSV. For this purpose, primers and probes are designed for the same targeted region of gG of VHSV. The ssRNA standards were prepared to find the detection limits of the assay. Detection limits were found ten-fold differences between real-time RT-PCR and real-time RT-RPA. While the detection limit of the RT-PCR was found as 95.5 viral RNA molecules/reaction in 95% probit value, the detection limit of RT-RPA was found as 943.75 viral RNA molecules/reaction in 95% probit value using ssRNA standards. These results show that RPA is a suitable test for VHSV Ie detection.
鱼类病毒性疾病给水产养殖业造成了巨大的经济损失。病毒性出血性败血症病毒（VHSV）是影响80多种鱼类的最重要的病毒性疾病之一。对该疾病的检测，尤其是在野外的检测，对于管理疾病预防和控制计划至关重要。重组酶聚合酶扩增（RPA）是一种等温方法，扩增时间非常短，单一孵育温度为37至42°C，是聚合酶链反应（PCR）的良好替代方法。本研究旨在开发一种与实时RT-PCR一样灵敏的RPA检测方法来检测VHSV。为此，针对VHSV的gG相同靶向区域设计了引物和探针。制备了单链RNA标准品以确定该检测方法的检测限。发现实时RT-PCR和实时RT-RPA的检测限相差十倍。在95%概率值下，RT-PCR的检测限为95.5个病毒RNA分子/反应，而使用单链RNA标准品时，RT-RPA在95%概率值下的检测限为943.75个病毒RNA分子/反应。这些结果表明RPA是检测VHSV Ie的合适检测方法。
