Development of a duplex real-time PCR assay for detection of Vibrio parahaemolyticus and Vibrio vulnificus

Objective To develop a duplex real-time PCR assay for detection of Vibrio parahaemolyticus and Vibrio vulnificus. Methods The sequences of tdh and vvh A genes were searched and blasted, primers and probes of these two genes were designed. The annealing temperature and the concentration of primers, probes, Mg2+, Taq DNA polymerase and d NTPs were optimized for detection. The specificity and sensitivity of the new method were analyzed. The simulative samples were tested. Results A real-time method for detection of V. parahaemolyticus and V. vulnificus was developed. This method showed good specificity and the detection limit was 103 CFU / ml. Conclusion This duplex real-time PCR assay can rapidly, sensitively and specifically identify V. parahaemolyticus and V. vulnificus.