Simultaneous detection of five pathogenic Vibrio species in seafood by a multiplex polymerase chain reaction coupled with high performance liquid chromatography assay

摘要

A multiplex polymerase chain reaction-based procedure followed by high performance liquid chromatography (mPCR-HPLC) assay was developed for the simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus and Vibrio mimicus from seafood. A set of multiplex PCR primers was designed based on the dnaJ gene of Vibrio species and was employed in a multiplex PCR procedure, then PCR products were subjected to HPLC analysis with PS-DVB-C18 particles DNASep column in which the unique HPLC profile generated from each specific amplicon represented corresponding Vibrio species. Our optimized mPCR-HPLC assay showed a higher sensitivity (ca. 10(1) CFLT/mL (g) pure culture or food homogenate) than that of gel electrophoresis technique. Specificity of the mPCR-HPLC assay was tested on a total of 227 strains of Vibrio species and 89 non-Vibrio species. Specific HPLC peak profiles were produced in strains belonging to the target Vibrio species, but not in all strains other than the five species, showing high specificity of the assay. A total of 7255 seafood samples were collected to evaluate diagnostic capability of the mPCR-HPLC assay and results showed that 339 samples were positive in this assay, in accordance with the testing results of conventional culture-, biochemical-based assays. Results indicated that the mPCR-HPLC assay developed in this study was applicable for simultaneous detection of the five pathogenic Vibrio species from seafood in routine laboratories, providing a useful supplement to conventional methods for routine monitoring and risk assessment of seafood. (C) 2015 Elsevier Ltd. All rights reserved.