Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups

Abstract

To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V. cholerae) serogroups non-O1 and non-O139. The outer membrane protein gene (ompW) specific for V. cholerae, as well as O antigen rfb genes specific for both O1 and O139, were used for the design of the PCR primers. Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139. Specific rfb genes and ompW were developed to evaluate their specificity, limit of detection, reproducibility and consistency. We established multiple PCR and real-time SYBR green PCR methods. According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR), both methods could specifically detect the non-O1, non-O139 V. cholerae, and to differentiate them from O1,O139 V. cholerae, other five Vibrios and 3 intestinal bacteria. The detection limits were 7 × 10(4) cfu/ml (multiple PCR) and 7 × 10² cfu/ml(real-time SYBR green PCR), with statistically significant difference seen (P < 0.05). For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92% while the internal coefficient variation ranging from 0.27% to 1.41%. 370 strains of non-O1, non-O139 V. cholerae, were detected, with both consistency rates as 100% . Both multiple PCR and real-time SYBR green PCR could detect non-O1, non-O139 V. cholerae, rapidly, specifically, and reproducibly, that could all be used for the detection and identification of non-O1, non-O139 under different conditions.