3种致病菌多重real-time PCR检测方法的建立及其在散装即食肉制品中的应用

建立一种同时快速检测沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌的三重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法.以沙门氏菌invA基因、金黄色葡萄球菌Sa442基因和蜡样芽孢杆菌Cereolysin AB基因为靶基因设计引物和TaqMan探针...

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.