Abstract:The lolB gene has been shown to code an outermembrane lipoprotein, and serve as a reliable molecular marker for the detection of all V.cholerae serogroup and biotypes. A pair of specific primers based on lolB gene of V.cholerae was designed, and a real-time PCR using SYBR Green I for V.cholerae detection was established. A 519bp gene fragment was amplified from chromosomal DNA of V.cholerae, and no positive reaction was detected in 3 other pathogenic Vibrio species using conventional PCR. The melting curve analysis of SYBR GreenⅠreal-time PCR showed one specific peak with melting temperature（Tm）of 86.5~87 ℃, and no primer-dimers peak present. The results indicated that the PCR primers have good specificity. Amplification curves of SYBR GreenⅠreal-time PCR revealed the geometric phase and plateau phase of PCR. Analysis of standard curves revealed excellent correlation between the quantity of bacteria (2.59×108 to 2.59×100) and PCR threshold cycle (Ct),with the correlation rate of 0.993. The results indicated that the SYBR GreenⅠreal-time PCR could be used as an effective assay for quantification of pathogenic V.cholerae. The assay could be completed within 4~5 h from extraction of nucleic acids to analysis of results. The SYBR GreenⅠreal-time PCR was a simple, rapid, specific and sensitive method for the diagnosis of aquatic animal diseases and investigation of epidemics caused by V.cholerae.
