Establishment and Application of Multiple Real-time PCR Method for Detection of Three Kinds of Pathogenic Bacteria in Bulk Ready-to-eat Meat Products

A triplex real-time polymerase chain reaction (real-time PCR) method for rapid simultaneous detection of Salmonella (SAL), Staphylococcus aureus (SA) and Bacillus cereus (BC) was established. Primers and TaqMan probes were designed targeting the invA gene of SAL, the Sa442 gene of SA, and the Cereolysin AB gene of BC. The performance indicators of the developed method were evaluated, and actual samples of ready-to-eat meat products were detected by it. The results showed that the real-time PCR method was highly reproducible, specific and sensitive. Fifteen non-target reference strains were tested by this method and the results were all negative. The standard curves for the three pathogenic bacteria were linear over the concentration range of 10²-10⁸ CFU/mL, and the intra-assay and inter-assay coefficients of variation (CVs) were less than 2% in the quantitative tests. Without bacterial enrichment, the limits of detection (LOD) for SAL, SA, and BC were 3.8 × 10², 4.9 × 10², and 5.7 × 10² CFU/mL, respectively. After 5 h enrichment, they increased to 3.8, 4.9, and 5.7 CFU/mL, respectively. The results of the real-time PCR method for 100 actual samples were consistent with those of the national standard method, indicating that the real-time PCR method can be used for the detection of the three pathogenic bacteria in bulk ready-to-eat meat products.