Objective: Targeting the specific O-antigen gene cluster, a Taq-Man real-time fluorescence PCR assay was developed to detect 01 and O139 Vibrio cholera concurrently and avoid frequently occurred false positives.
Methods: Two pairs of specific primers and two Taq-Man fluorescent probes respectively targeting rfbM gene of Vibrio cholerae O1 and wbfR of 0139 were designed. After optimization of conditions, the specialty and sensitivity of the detection method were evaluated and ten simulated food samples and 128 clinical samples were tested.
Results: The detection limits of Vibrio cholerae O1 and O139 were 92 CFU/ml and 116 CFU/ml, which were almost the same with the usual PCR method. The developed real-time fluorescence PCR protocol detected only Vibrio cholerae O1 and O139 and it was not affected by many normal food pathogens such as Staphylococcus aureus and Salmonella, especially Vibrio cholerae O141 which contained TCP genes and Vibrio mimicus which contained ZOT gene. The clinical detect results of the developed assay and routine culture method were exactly the same.
Conclusion: The developed detection assay could quantitatively detect Vibrio cholerae O1 and O139 concurrently in only 3 hours, and could avoid false positive, and thus was an efficacious method for detecting Vibrio cholerae O1 and O139 and could be used in a wide area such as entry-exit inspection and quarantine, food safety detection and clinical diagnose.