Multiplex Polymerase Chain Reaction Assay for Rapid Detection of Three Foodborne Pathogens in Low-Temperature Meat Products
LI Xinfu1,2, WANG Zhouping1, LI Cong1,2, FANG Lingli1, ZHAO Ying2, XU Baocai1,2,3,*
1.School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; 2.The State Key Laboratory of Meat Processing and Quality Control, Jiangsu Yurun Meat and Food Co. Ltd., Nanjing 211806, China; 3.Jiangsu Collaborative Innovation Center of Meat Productionand Processing, Quality and Safety Control, Nanjing 210095, China
Abstract：Salmonella spp., Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) are 3 species of bacteria which can easily contaminate low-temperature meat products. The objective of this study was to develop a rapid and efficient method for the simultaneous detection of the bacterial pathogens using multiplex polymerase chain reaction(m-PCR). The specific primers for the invA gene of Salmonella spp., the PrfA gene of L. monocytogenes and the nuc gene of S.aureus were designed and their specificity was determined. After enrichment culture, the 3 strains were inoculated to lowtemperature meat products and incubated overnight for evaluating the sensitivity of the detection method. Optimization of the PCR system and its application to the detection of real samples were carried out. Our experimental results showed that the optimum PCR conditions were determined as follows: using a reaction system containing 2.5 μL of 10 × PCR buffer,2.0 μL of 2.5 mmol/L dNTPs, 0.5 μL of 2.5 U/μL DNA polymerase, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1.0 μL of L. monocytogenes template, 1.0 μL of S. aureus template or 2.0 μL of Salmonella spp. template brought with ddH2O up to 25 μL, and an annealing temperature of 64 ℃. The multiplex PCR method was applicable to detect Salmonella spp. in meat products with the advantages of rapidity, high sensitivity and specificity and simplicity.
