Rapid and sensitive detection of pathogenic Elizabethkingia miricola in black spotted frog by RPA-LFD and fluorescent probe-based RPA

•Established fluorescent probe-based RPA (exo RPA) and RPA-LFD methods for fast and sensitive detection of E. miricola.•Exo RPA and RPA-LFD could detect E. miricola genomic DNA at 38 °C in 30 min.•The detection sensitivity of exo RPA and RPA-LFD were 102 copies/μL.•The exo RPA and RPA-LFD achieved rapid detection of E. miricola without bulky lab equipment. Elizabethkingia miricola 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 E. miricola, which can accomplish the examination at 38 °C within 30 min. The limiting sensitivity of RPA-LFD and exo RPA (102 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 (E. miricola, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas veronii, CyHV-2 and Edwardsiella ictaluri), and the result showed that the single band was displayed in E. miricola 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 E. miricola accurately and rapidly. In summary, the methods of RPA-LFD and exo RPA were able to detect E. miricola conveniently, rapidly, accurately and sensitively. This study provides prospective methods to detect E. miricola infection in frog culture.