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目的建立食源性金黄色葡萄球菌杀白细胞毒素luk-F、luk-S和葡萄球菌A蛋白SPA基因的多重PCR检测方法。方法设计luk-F、luk-S和SPA基因的引物,利用单重PCR方法和测序验证引物的特异性;建立多重PCR快速检测方法,并对方法的特异性进行评价。结果利用单对引物对实验室的多个菌株进行盲筛,出现的条带均为单一条带,且与目的片段长度一致。多重PCR反应体系:25μl套装反应液2,上、下游引物(20μmol/L)各0.4μl,0.25μl套装反应液1,基因组模板6μl,加超纯水至50μl。扩增条件:94℃预热2 min;94℃保持30 s,54℃保持60 s,72℃保持60 s,34个循环;72℃延伸5 min。结论该方法对金黄色葡萄球菌有很好的特异性,快速简便,可以同时对大量菌株进行筛选。
Objective To establish a multiplex PCR method for the detection of SPA gene of food-borne Staphylococcus aureus leukotoxin luk-F, luk-S and staphylococcal protein A. Methods The primers of luk-F, luk-S and SPA genes were designed. The specificity of the primers was verified by single-PCR and sequencing. The multiplex PCR method was established and the specificity of the method was evaluated. Results A single pair of primers was used for blind screening of several strains in the laboratory. The bands appeared were single band and consistent with the length of the target fragment. Multiplex PCR reaction system: 25μl set reaction solution 2, 0.4μl each of the upper and lower primers (20μmol / L), 0.25μl set reaction solution 1, 6μl genomic template, and ultrapure water to 50μl. Amplification conditions: 94 ℃ preheated 2 min; 94 ℃ for 30 s, 54 ℃ for 60 s, 72 ℃ for 60 s, 34 cycles; 72 ℃ for 5 min. Conclusion This method has a good specificity for Staphylococcus aureus, fast and easy, and can be screened for a large number of strains at the same time.