Multiplex real-time TaqMan-based PCR method for rapid detection of toxigenic Vibrio cholerae Serogroup O139 in simulated clinical specimens

Objective:To establish a TaqMan-based multiplex real-time PCR assay for rapid detection of toxigenic Vibrio cholerae Serogroup O139 in simulated clinical specimens.Methods:The gene sequences of Cholera toxin and specific O antigen biosynthetic gene rfb O139 Serogroup were aligned using the biologic software and the specific primers and probe were designed in the conserved region of the CT and rfb O139 gene for Vibrio cholerae serogroup O139.The reaction conditions were optimized and the sensitivity,specificity of the assay were evaluated.The simulated clinical specimens prepard from the fecal and the environment were detected by this assay.Results:For specific detecting the toxigenic Vibrio cholera Serogroup O139,the detection limits of the assay for rfb O139 and CT gene all were 100 cfu/ml(or 5 cfu/PCR reaction).The result of detecting the simulated clinical specimens prepard from the fecal and the environment was in accord with actual situation.It took only three hours to do the multiplex real-time PCR.Conclusion:This assay is a rapid,sensitive and specific one for the detection of toxigenic Vibrio cholerae Serogroup O139 from fecal and environment specimens.