Rapid and sensitive detection of mud crab Scylla serrata reovirus by a reverse transcription loop-mediated isothermal amplification assay

Abstract

Scylla serrata reovirus (SsRV) is one of the most prevalent viral pathogens of the mud crab (S. serrata). This pathogen is widespread in east China and causes severe economic losses to the nation's mud crab industry. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In the present study, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and sensitive detection of SsRV was developed and evaluated. The LAMP reaction mix was optimized, as were the reaction temperature (62°C) and the duration of the assay (60min). The sensitivity of the RT-LAMP assay was determined to be 0.8fg SsRV dsRNA, which was 1000-fold higher than that of a one-step reverse transcription polymerase chain reaction (RT-PCR). The RT-LAMP assay also had higher sensitivity than a one-step RT-PCR, as it identified nine more positive cases from 55 mud crabs suspected of having SsRV. No cross-reactivity was found with the DNA/RNA of other tested viruses and SsRV-negative animals. Importantly, the assay can be completed within 60min and is faster than conventional RT-PCR. In summary, the RT-LAMP assay is a simple, cost-effective, sensitive, and specific tool for the rapid detection of SsRV infection.