A Rapid and Sensitive Method for Simultaneous Screening of Nine Foodborne Pathogens Using High-Performance Liquid Chromatography Assay

Abstract

We developed a multiplex PCR-based procedure followed by high-performance liquid chromatography (mPCR-HPLC) assay for high-throughput screening foodborne pathogens, including Salmonella spp., Listeria monocytogenes, Enterobacter sakazakii, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus. Vibrio species-specific primers were designed targeting dnaJ gene, and pathogen-specific primers for L. monocytogenes, Salmonella spp., E. sakazakii, E. coli O157:H7, Shigella spp., and S. aureus were designed targeting iap, fimY, 16S ribosomal RNA (rRNA), rfbE, ipaH, and 442 genes, respectively. PCR products were analyzed using WAVE-4500 DNA system equipped with PS-DVB-C18 particles DNASep column and each specific amplicon generated a characteristic chromatographic profile. Detection limit of mPCR-HPLC assay was ca. 101 CFU/mL in pure cultures and less than 102 CFU/g in contaminated matrixes. A total of 395 bacterial strains were used for specificity testing of the mPCR-HPLC assay, and specific HPLC profiles were only produced in strains belonging to the target, showing a high specificity. Applying the assay to 2677 samples collected from clinical, food, and environmental sources revealed that 917 samples were positive, in accordance with bacterial isolation. The high sensitivity and specificity of mPCR-HPLC assay indicate its great potential to be a powerful tool for high-throughput screening foodborne pathogens.