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A Rapid Multiplex PCR‐DHPLC Method of Detection and Identification of Pathogenic Bacteria in Aquatic Products

Zhan, Xiaowei, Zheng, Qiuyue, Fu, Junfan, Xu, Junyi, Cao, Jijuan
Journal of food safety 2015 v.35 no.1 pp. 50-58
DNA primers, Listeria monocytogenes, Vibrio cholerae, bacteria, genes, high performance liquid chromatography, pathogens, polymerase chain reaction, rapid methods
In this study, we established a multiplex polymerase chain reaction‐denaturing high‐performance liquid chromatography (MPCR‐DHPLC) method for rapid detection of the aquatic‐associated pathogens Vibrio cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and Listeria monocytogenes. Specific primer sets targeting the dnaJ gene of the Vibrio species and the hly gene of L. monocytogenes were used. We also defined the optimal primer concentrations to detect each Vibrio species. We used the MPCR‐DHPLC method to identify target bacteria in aquatic products. Performance characteristics of the MPCR‐DHPLC system showed that it is specific for six common pathogenic bacterial species and can be used for the practical detection of these pathogens in aquatic products. Furthermore, two practical applications of two environmental water samples were tested according to this method by MPCR‐DHPLC, then the results of detection were consistent with conventional tests but faster. It showed that this method proved to be a fast, sensitive and specific tool for V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and L. monocytogenes detection in a routine microbiological laboratory. PRACTICAL APPLICATIONS: Various bacterial species can be simultaneously detected with the same PCR reaction. A MPCR‐DHPLC method of identification of various bacterial species was established. This method improves test efficiency and minimizes test time.