Rapid and Sensitive Detection of Pathogenic in Black Spotted Frog by RPA-LFD and Fluorescent Probe-based RPA

is a highly infectious pathogen, which causes high mortality rate in frog farming. Therefore, it is urgent to develop a rapid and sensitive detection method. In this study, two rapid and specific methods including recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and fluorescent probe-based recombinase polymerase amplification (exo RPA) were established to effectively detect , which can accomplish the examination at 38 °C within 30 min. The limiting sensitivity of RPA-LFD and exo RPA (10 copies/μL) was ten-fold higher than that in generic PCR assay. The specificities of the two methods were verified by detecting multiple DNA samples (, CyHV-2 and ), and the result showed that the single band was displayed in DNA only. By tissue bacterial load and qRT-PCR assays, brain is the most sensitive tissue. Random 24 black spotted frog brain samples from farms were tested by generic PCR, basic RPA, RPA-LFD and exo RPA assays, and the results showed that RPA-LFD and exo RPA methods were able to detect accurately and rapidly. In summary, the methods of RPA-LFD and exo RPA were able to detect conveniently, rapidly, accurately and sensitively. This study provides prospective methods to detect infection in frog culture.