Development of duplex real-time TaqMan PCR assay for detection of specific gene sequence from toxigenic Vibrio cholerae serogroup O139

Objective:To develop a sensitive and specific duplex TaqMan real-time quantitative polymerase chain reaction(PCR) assay for the detection of toxigenic Vibrio cholerae O139.Methods:Designed primers and TaqMan probes for duplex PCR reaction using specific O antigen biosynthetic gene rfb-o139 of serogroup O139 and cholera toxin gene ctxA as the target regions for amplification.Smart cycler system was used to evaluation the sensitivity of constructed assay method,and totally 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection were also used to measure the specifity of constructed method.Results:The sensitivity of constructed assay method was 102 copies per reaction for the testing of rfb-O139 gene.Using the Vibrio cholerae O139 genome DNA as the starting materials for detection,constructed assay method can detect as low as 1.0×100 pg per reaction.Non-specific amplifications were not presented when testing 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection.In addition,the assay could get valid results within only 2 hours.Conclusion:The duplex TaqMan real-time PCR assay described in this paper was so specific,sensitive and rapid that it was highly suitable not only for the discrimination of detection of Vibrio cholerae serogroup O139,but also for the detection of its virulent abilities simultaneously.