应用多重PCR快速鉴定自然发酵肉制品中6种葡萄球菌

Rapid Identification of Six Staphylococcus Species in Fermented Meat Products Using Multiplex

肉葡萄球菌(Staphylococcuscarnosus)、木糖葡萄球菌(S.xylosus)、腐生葡萄球菌(S. saprophyticus)、表皮葡萄球菌(S. epidermidis)、马胃葡萄球菌(S. equorum)和松鼠葡萄球菌(S. sciuri)是发酵肉制品中常见的葡萄球菌,为准确、快速地检测和鉴定这些菌种,建立6种葡萄球菌的多重聚合酶链式反应(polymerase chain reaction,PCR)方法并进行验证。以上述菌种的gap、rpoB、SodA和SNc为靶基因,设计和筛选菌种特异性引物6对,优化PCR体系,建立6种葡萄球菌的多重PCR鉴定方法。实验结果显示多重PCR的基因组DNA检测灵敏度可达到4 pg,活菌检测灵敏度可达到2×10~2 CFU;采用建立的多重PCR方法对传统肉制品中分离的8株葡萄球菌进行鉴定,结果与16S rDNA序列分析、生理生化鉴定结果一致。建立的多重PCR方法具有较高的种间特异性,可快速、准确地用于发酵肉制品中肉葡萄球菌、木糖葡萄球菌、表皮葡萄球菌、马胃葡萄球菌、松鼠葡萄球菌和腐生葡萄球菌的检测和鉴定,具有较好的应用前景。

A multiplex polymerase chain reaction(mPCR)assay was developed and validated for simultaneous detection of Staphylococcus carnosus,S.xylosus,S.saprophyticus,S.epidermidis,S.equorum and S.sciuri,which are commonly found in fermented meat products.In this study,six pairs of species-specific primers were designed targeting the gap,rpoB,SodA and SNc genes,and the PCR reaction system was optimized.The results showed that The sensitivity of the mPCR system was4pg for bacterial genomic DNA and2×10^2CFU for living cultures.Meanwhile,eight Staphylococcus strains from traditional fermented meat products were consistently identified by this mPCR assay and16S rDNA sequence and biochemical analysis.This mPCR method was species-specific and effective,and could be used in the rapid and accurate identification of S.carnosus,S.xylosus,S.epidermidis,S.equorum,S.sciuri and S.saprophyticus.Thus,it has great application prospects.