Real-time quantitative isothermal detection of Ostreid herpesvirus-1 DNA in Scapharca subcrenata using recombinase polymerase amplification.

Ostreid herpesvirus-1 (OsHV-1) is a well-known pathogen associated with high mortality rates in hatchery-reared larvae and juveniles of different bivalve species worldwide. Early, rapid and accurate diagnosis plays a fundamental role in disease prevention and control in aquaculture. Recombinase polymerase amplification (RPA) is a novel isothermal amplification method, which can amplify detectable amount of DNA at 37 °C-39 °C within 20 min. In the present study, two sets of specific primers and probes were designed for the real-time quantitative RPA (qRPA) detection of OsHV-1 DNA. The sensitivity and specificity of detection were evaluated by comparison with quantitative polymerase chain reaction (qPCR). The detection limit for qRPA assays was shown to be 5 copies DNA/reaction for the primer set ORF95, which was lower than the 100 copies required for the qPCR test. The optimal reaction temperature and time were 37 °C for 20 min, making this approach faster than qPCR. This is the first study to apply qPCR and qRPA methods to detect OsHV-1 in Scapharca subcrenata. The percentage of viral load sample detected by the two methods was 22% and the correlation of the two virus quantitative results was 0.8. Therefore, qRPA assays is sensitive, fast, and high-temperature independent relative to qPCR and is suitable for critical clinical diagnostics use and rapid field analysis in resource-limited settings.