RPA-LFD coupled with triple-target gene analysis for on-site detection of Vibrio vulnificus and Photobacterium damselae

Vibrio vulnificus and Photobacterium damselae , zoonotic Vibrio pathogens, pose dual threats to human and marine animal health. The development of rapid and precise detection methods for these bacteria remains a critical unmet need in public health surveillance and aquatic food safety control. This study firstly used gradient salting-out combined with SDS-PAGE and HPLC-MS/MS (High Performance Liquid Chromatography-Tandem Mass Spectrometry) to screen out stable specific expressed genes in these two Vibrio pathogens as detecting targets. Three specific targets ( RsxG - HutX - DsbC in V. vulnificus or HflD - McP - Pl8F in P. damselae ) were obtained and further used to develop rapid on-site detection methods based on RPA-LFD (Recombinase polymerase amplification intergrated lateral flow dipstick). The detection method showed good specificity and had no cross-reactivity with the other parallely tested seven common aquatic bacteria. The whole detection process could be completed within 20 min. The lowest detection limit for the pure culture of V. vulnificus and P. damselae was 3×10 1 CFU/mL and 5×10 1 CFU/mL. The successful detection of V. vulnificus and P. damselae in the artificially contaminated seafood samples validated their universal applicability in aquatic product. In conclusion, the developed RPA-LFD method for on-site detection of V. vulnificus and P. damselae was sensitive and specific, and it is of critical importance for food safety emergency response and health aquaculture. • Novel triple-gene targets for Vibrio vulnificus ( RsxG - HutX - DsbC ) and Photobacterium damselae ( HflD - McP - Pl8F ) were identified. • A rapid (under 20 min) RPA-LFD assay was developed for on-site, visual detection of V. vulnificus and P. damselae. • The RPA-LFD method demonstrated high sensitivity (3×10 1 CFU/mL for V. vulnificus , 5×10 1 CFU/mL for P. damselae ) and specificity.