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目的利用实时荧光定量-聚合酶链反应(real-time PCR)技术,建立食品中金黄色葡萄球菌(金葡萄)污染的快速敏感特异的检测方法。方法以金葡菌的FemB基因作为靶序列,设计一对引物和探针,以金葡菌菌株提取核酸DNA作为模板,优化引物和探针的浓度比和Mg2+浓度,以金葡菌和10种相关细菌考核检测体系的灵敏性、稳定性和特异性。并初步应用于样品的检测。结果本研究建立的反应体系在引物和探针的浓度为0.8μmol/L、0.6μmol/L,Mg2+浓度为3.5 mmol/L时,具有良好的特异性和敏感性。在10株相关菌株的检测中,除金葡菌出现很好的阳性外,其余菌株均为阴性。在纯菌条件下,最低检测限为44 cfu/m。l稳定性分析表明:同一样品重复检测3次Ct值的变异系数均<5%。检测样品结果显示real-tim e PCR方法较传统方法敏感、快捷、简便。结论该方法特异性强,稳定性高,操作简便快捷,适应食品微生物检验发展需要,具有较大的推广及应用价值。
Objective To establish a rapid and sensitive method for the detection of Staphylococcus aureus (GOLD) contamination in food by real-time fluorescence quantitative-polymerase chain reaction (real-time PCR). Methods A pair of primers and probes were designed based on the FemB gene of Staphylococcus aureus. DNA was extracted from Staphylococcus aureus as a template to optimize the concentration ratio of primers and probes and the concentration of Mg2 +. Staphylococcus aureus and 10 The sensitivity, stability and specificity of the relevant bacterial test system. And initially applied to the detection of samples. Results The reaction system established in this study had good specificity and sensitivity when the concentration of primer and probe was 0.8 μmol / L, 0.6 μmol / L and Mg2 + concentration was 3.5 mmol / L. In the detection of 10 related strains, all strains were negative except for S. aureus. Under pure conditions, the minimum detection limit is 44 cfu / m. l The stability analysis showed that the coefficient of variation (CV) of Ct value of the same sample was 3% less than 5%. Test sample results show that real-tim e PCR method is more sensitive than traditional methods, fast and easy. Conclusion The method is of high specificity, high stability, simple and quick operation, and adaptability to the development of food microbiology test, which has great promotion and application value.