The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System

Abstract

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: <i>toxS</i> for <i>Vibrio parahaemolyticus</i>, <i>virR</i> for <i>Listeria monocytogenes</i>, <i>recN</i> for <i>Cronobacter sakazakii</i>, <i>ipaH</i> for <i>Shigella flexneri</i>, <i>CarA</i> for <i>Pseudomonas putida</i>, <i>rfbE</i> for <i>Escherichia coli</i>, <i>vvhA</i> for <i>Vibrio vulnificus</i>, and <i>gyrB</i> for <i>Vibrio alginolyticus</i>. 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 <i>V. parahaemolyticus</i>, <i>L. monocytogenes</i>, <i>E. coli</i> O157:H7, <i>C. sakazakii</i>, <i>S. flexneri</i>, <i>P. putida</i>, <i>V. vulnificus</i>, and <i>V. alginolyticus</i>, 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.