Development of a rapid triplex TaqMan real-time PCR assay for the detection of toxigenic Vibrio cholerae

Abstract

Objective To develop a rapid triplex TaqMan real-time quantitative polymerase chain reaction (PCR) assay for the discrimination of toxigenic Vibrio cholerae O1 and Vibrio cholerae O139 simultaneously. Methods Primers and Taqman probes were designed for multiplex PCR reaction using rfb-O1, rfb-O139 specific O antigen biosynthetic gene and cholera toxin gene ctxA as the target regions for amplification. Smart cycler system was used to evaluate the sensitivity of this method, and a total of 19 other common enteropathogenic bacteria or frequent isolates causing nosocomial infection were also used to measure the specificity of constructed method. Results Constructed assay method allowed the detection as few as 102 copies per reaction for the testing of ctxA, rfb-O1 and rfb-O139 genes using mixed plasmids as the templates, and the linearity range of assay varied from 102 to 109 copies per reaction. Using the Vibrio cholerae O1 and O139 genome DNAs as the starting materials for detection, constructed assay method could detect 1.0×10-1 pg and 1.0×100 pg per reaction,respectively. Nan-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 triplex TaqMan real-time PCR assay described in this study was specific, sensitive,rapid and suitable not only for the discrimination of toxigenic strains,O1 and O139 serogroup of Vibrio cholerae, but also for the detection of their virulent abilities simultaneously. Key words: Vibrio cholerae; Polymerase chain reaction; Taq polymerase; Cholera toxin