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目的建立菌液SYBR荧光PCR法快速检测金黄色葡萄球菌的方法。方法根据金黄色葡萄球菌的特异基因耐热核酸酶nuc设计引物,PCR方法和SYBR荧光PCR溶解曲线法验证引物的特异性;建立菌液SYBR荧光PCR快速检测方法,奶粉加标实验和菌株特异性实验评价方法的特异性,制备基因组浓度梯度和菌落梯度检验方法的灵敏性。结果利用引物对实验室的3个标准菌株进行PCR扩增,出现的条带均为单一条带,且与目的片段长度一致。16株金黄色葡萄球菌菌株的熔解曲线均呈单峰,特异性良好,无非特异性扩增。建立的菌液SYBR荧光PCR方法对金黄色葡萄球菌有很好的特异性和灵敏性,灵敏度达1 pg/μl DNA浓度,并可检测1 CFU/ml的复杂样品。结论本研究建立的菌液SYBR荧光PCR方法快速、特异性好、灵敏性高,对快速检测样品中金黄色葡萄球菌有重要意义。
Objective To establish a rapid SYBR fluorescence PCR method for the detection of Staphylococcus aureus. Methods Primers were designed according to nuclease nuc primers of specific gene of Staphylococcus aureus, and the specificity of the primers was verified by PCR and SYBR fluorescence PCR. The rapid detection method of SYBR fluorescence PCR, The specificity of the experimental evaluation method, the sensitivity to prepare the genomic concentration gradient and the colony gradient test method. Results PCR amplification was carried out on 3 standard strains in the laboratory using primers. The bands appeared were single band and consistent with the length of the target fragment. The melting curves of 16 strains of Staphylococcus aureus showed a single peak with good specificity and no nonspecific amplification. The established SYBR Fluorescent PCR assay for S. aureus is very specific and sensitive, with a sensitivity of 1 pg / μl DNA concentration and can detect complex samples at 1 CFU / ml. Conclusion The SYBR fluorescence PCR method established in this study is rapid, specific and sensitive. It is of great significance for rapid detection of Staphylococcus aureus in samples.