real-time recombinase polymerase amplification assay for the detection of vibrio cholerae in seafood

Vibrio cholerae, the most hazardous vibrio species, is threatening aquacultured animals and even human beings. in recent years, a few genetic markers that target v. cholerae have been reported, but their application was limited by techniques or themselves. the outer membrane lipoprotein gene lolb has been identified as a specific marker by using pcr and quantitative pcr methods. in this study, we developed a real-time recombinase polymerase amplification (rpa) assay targeting lolb gene in an attempt to provide a sensitive, rapid, and reliable detection method of v. cholerae. the sensitivity of rpa assay was determined by amplifying the standard plasmid dilutions. the detection limit down to five copies was achieved within 10 min, which was lower than that of qpcr (ten copies after 25 cycles within 1.5 h). the reproducibility of rpa assay was verified by eight independent experiments, presenting a high r 2 value of the standard regression line (0.97). in addition, the specificity was confirmed with dna extracted from other bacteria, shrimps, clams, and fishes using both methods. finally, v. cholerae in shrimp samples were quantified using real-time rpa and qpcr with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. the applicability in the food industry was confirmed by quantitative detection of natural seafood.