Multiplex polymerase chain reaction (PCR) assays are mainly used to simultaneously detect or identify multiple pathogenic microorganisms. To achieve high specificity for detecting foodborne pathogenic bacteria, specific primers need to be designed for the target strains. In this study, we designed and achieved a multiplex PCR system for detecting eight foodborne pathogenic bacteria using specific genes: toxS for Vibrio parahaemolyticus, virR for Listeria monocytogenes, recN for Cronobacter sakazakii, ipaH for Shigella flexneri, CarA for Pseudomonas putida, rfbE for Escherichia coli, vvhA for Vibrio vulnificus, and gyrB for Vibrio alginolyticus. The sensitivity of the single system in this study was found to be 20, 1.5, 15, 15, 13, 14, 17, and 1.8 pg for V. parahaemolyticus, L. monocytogenes, E. coli O157:H7, C. sakazakii, S. flexneri, P. putida, V. vulnificus, and V. alginolyticus, respectively. The minimum detection limit of the multiplex system reaches pg/μL detection level; in addition, the multiplex system exhibited good specificity and stability. Finally, the assays maintained good specificity and sensitivity of 10⁴ CFU/mL for most of the samples and we used 176 samples of eight aquatic foods, which were artificially contaminated to simulate the detection of real samples. In conclusion, the multiplex PCR method is stable, specific, sensitive, and time-efficient. Moreover, the method is well suited for contamination detection in these eight aquatic foods and can rapidly detect pathogenic microorganisms.

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