利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

Research on degenerate primers for detection of Staphylococcus aureus enterotoxin A,B gene

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。

The degenerate primers were designed to establish a PCR assay for synchronous and fast detection of staphylococcal enterotoxin A and B.According to the sequences of SEA and SEB gene,primers were designed and used for PCR.The lengths of the amplified primers were 105 bp and 135 bp.To evaluate the specificity of PCR assay,2strains of SEA,SEB and 4strains of non-SEA-B were tested by PCR.SEB was 10-fold serially diluted and amplified by PCR to verify its sensitivity.The results showed that the detection of Staphylococcus aureus enterotoxin A,B strain DNA results were positive and 4strains of control strains tested negative.The specificity of PCR products was confirmed by DNA sequencing.In addition,the minimum detection limit of DNA was 3.58 ng of SEB and the whole process was less than 20 h.A rapid,specific and sensitive detection method was established and the method can simultaneous detect Staphylococcal enterotoxins A,B in the same reaction condition.