Development of a rapid duplex TaqMan real-time PCR assay for detection of specific gene sequence from toxigenic Vibrio cholerae serogroup O1

Objective To develop a sensitive and specific duplex TaqMan real-time quantitative polymerase chain reaction(PCR) assay for detection of toxigenic Vibrio cholerae O1.Methods Designed primers and Taqman probes were used for duplex PCR reaction using specific O antigen biosynthetic gene rfb of O1 serogroup and cholera toxin gene ctxA as the target regions for amplification.Smart cycler system was used to evaluate the sensitivity of constructed assay method and a total of 19 other common enteropathogenic bacteria or common isolates causing nosocomial infection were also used to measure the specificity of constructed method.Results The sensitivity of constructed assay method was 102 copies per reaction for the test of rfb-O1 gene.Using the Vibrio cholerae O1 genome DNA as the starting materials for detection,the constructed assay method can detect as low as 1.0 × 10-1 pg per reaction.Non-specific amplifications were absent when testing 19 other common enteropathogenic bacteria or frequent nosocomial isolates.In addition,the assay could yield valid results only within 2 hours.Conclusion The duplex TaqMan real-time PCR assay described here is so specific,sensitive and rapid that it is perfectly suitable not only for the discriminative detection of O1 serogroup of Vibrio cholerae,but also for the detection of its virulence simultaneously.