Development of a recombinase polymerase amplification assay for viral haemorrhagic septicemia virus.

Abstract

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.