A multiplex polymerase chain reaction coupled with high-performance liquid chromatography assay for simultaneous detection of six foodborne pathogens

A multiplex polymerase chain reaction (PCR) coupled with high-performance liquid chromatography (HPLC) assay was developed to simultaneously detect Salmonella, Campylobacter jejuni, Listeria monocytogenes, Yersinia enterocolitica, Streptococcus hemolyticus and Staphylococcus aureus from foods. Six pairs of specific PCR primers were designed according to Salmonella invA, C. jejuni cdtA, L. monocytogenes pfrA, S. aureus femA, Y. enterocolitica 16S rRNA and St. hemolyticus cfb. Following the development of multiplex PCR, the PCR products were subjected to HPLC analysis. Unique HPLC peak profile for each PCR product represented corresponding bacterial strain, suggesting a better alternative to conventional PCR gel electrophoresis with ethidium bromide (EB) staining. The specificity analysis of the multiplex PCR-HPLC with 121 bacterial strains and 4 yeast strains showed that the method was highly specific for the target pathogens. One thousand and three hundred ninety-four blind samples were used to evaluate the practical diagnostic capability of the method, and results showed that eighty-eight food samples contaminated with single or multiple pathogens were detected by the method, which accorded with the testing results via conventional method. All data demonstrated that the multiplex PCR-HPLC is an efficient diagnostic method for rapid identification of the six foodborne pathogens. The simplicity and high sensitivity of the method may lead to improved management of food safety and foodborne diseases.

► A multiplex PCR-HPLC method for simultaneous detection of six foodborne pathogens was developed. ► The developed method suggests a better alternative to conventional PCR electrophoresis with EB staining. ►The developed method showed high sensitivity and specificity. ►The developed method may lead to improved management of food safety and foodborne disease.