[Development of multiplex real time PCR methodology for better identification and discrimination of Vibrio cholerae...

To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae.

Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3'end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens.

Vibrio cholerae was identified by multiplex real time PCR accurately and quickly, which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 10(2) cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays.

Our results showed that the multiplex real time PCR was a reliable, accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.