Real-time isothermal detection of Abalone herpes-like virus and red-spotted grouper nervous necrosis virus using recombinase polymerase amplification

Abstract

Abalone herpes-like virus (AbHV) and Red-spotted grouper nervous necrosis virus (RGNNV) are two serious viruses that infect animal populations in aquaculture. Both viruses cause diseases associated with high mortality rates, resulting in dramatic economic losses in the aquaculture industry. There are currently no effective treatments for either of these two viral diseases. Thus, early, rapid, and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Traditional methods of diagnosis, such as virus culture, enzyme-linked immunoassay, and polymerase chain reaction (PCR), are either time consuming or require sophisticated temperature control devices. In this study, one sets of specific primers and probes were designed for the real-time quantitative recombinase polymerase amplification (qRPA) detection of AbHV and RGNNV separately. The sensitivity and specificity of detection were evaluated by comparison with detection by conventional PCR and quantitative PCR. The optimal reaction temperature and time for virus detection is 37°C for 20min. The detection limit is 100 copies per reaction, making this approach faster and more sensitive than qPCR in this study. In a field application, the detection percentage of qRPA was higher than that of qPCR for both AbHV and NNV. Additionally, good correlation was found between qRPA and qPCR detection (R2>0.8). The methods presented here can be used as alternatives to qPCR for quick and quantitative detection of pathogens infecting aquaculture species.