Establishment of multiplex real-time quantitative PCR method for rapid detection of toxigenic Vibrio cholerae serogroup O1 from environmental sample

To establish a TaqMan-based multiplex real-time PCR assay for detection of toxigenic Vibrio cholerae serogroup O1 and construct a method for primary application of environmental samples test,the gene sequences of cholera toxin and specific O antigen biosynthetic gene rfb of serogroup O1 downloaded from the GenBank were aligned using the biologic software,and the specific primers and probe were designed in the conserved region of the CT and rfb-O1 gene for Vibrio cholerae serogroup O1.The reaction conditions were optimized and the sensitivity,specificity and the stability of the assay were evaluated.The clinical specimens collected from the environment were detected by this assay.For specifically detecting the toxigenic Vibrio cholerae serogroup O1,the detection limits of the assay for rfb-O1 and CT gene were 100 cfu/mL and the regression coefficient of the quantitative curve were 0.998 and 0.999,respectively.Five strains of toxigenic Vibrio cholerae serogroup O1 were collected from 352 environmental specimens for the first time by this assay.This assay is a rapid,sensitive and specific one for the widespread detection of toxigenic Vibrio cholerae serogroup O1 from environmental sample.