Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases.

Abstract

In this study, we describe a multiplex PCR method for the detection of five food-relevant virulence pathogenicity genes of intestinal pathogens. Five pairs of primers were designed based on nuc gene for Staphylococcus aureus, hlyA gene of Listeria monocytogenes, ipaH gene of Shigella flexneri, lysP gene of Yersinia enterocolitica and tpi gene of Clostridium difficile. Conditions were optimized to amplify fragments of those genes simultaneously in one PCR amplification. After developing and optimizing the multiplex PCR reaction system, the specificity and sensitivity of the multiple PCR assays were evaluated. The optimized program is also applied to retail meat for testing. The result indicated that when the annealing temperature was 54°C and the primer concentrations of S. aureus, L. monocytogenes, S. flexneri, Y. enterocolitica and C. difficile are 10, 10, 5, 3 and 2μM, the five strains could expand 484, 345, 204, 156, 88bp of clear fragments, respectively. So was the multiple PCR in artificially contaminated beef produce. All cultures were cultured and separated by traditional methods. The multiplex PCR method offers a rapid, simple, and accurate identification of pathogens and could be used in food safety investigations, clinical diagnosis as well as for the surveillance of the spreading determinants of pathogens in epidemiological studies.