多重PCR检测奶牛乳腺炎金黄色葡萄球菌粘附素基因clfa A、clfa B、fnbp A和fnbp B

Multiplex PCR to identify Staphylococcus aureus adhesins gene clfa A,clfa B,fnbp A and fnbpB involved in dairy cow mastitis

利用多重PCR检测金黄色葡萄球菌粘附素基因clfa A、clfa B、fnbp A和fnbp B的方法,对奶牛乳腺炎金黄色葡萄球菌临床分离株进行聚集因子主效基因的分析。通过设计合成的特异性引物对金黄色葡萄球菌模板进行PCR扩增,将目的基因回收并连接到T载体,鉴定后进行测序验证,然后对本实验室所分离鉴定的金葡菌临床分离株进行多重PCR检测。PCR产物经过电泳成像显示,clfa A和clfa B分别在292bp和205bp处出现特异性条带;fn-bp A和fnbp B分别在524bp和642bp处出现特异性条带。通过对29株金葡菌临床分离株多重PCR检测发现:能扩增出clfa A、clfa B、fnbp A和fnbp B的分别有26株、12株、28株和3株。建立的多重PCR检测金黄色葡萄球菌粘附素基因的方法具有良好的特异性和可靠性,并且发现clfa A和fnbp A基因存在于绝大部分的金黄色葡萄球菌中。

To establish a method for detection of Staphylococcus aureus adhesin gene clfa A, clfa B, fnbp A and fnbp B involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the target genes. Four pairs of oligonucleotide primers special to the sequence of target genes were designed according to the sequences which encode the Clfa and FnBP. DNA of the bacteria isolated from the milk of dairy cow with mastiffs was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The amplified gene was cloned into the pMD18-T plasmid, and the detected positive stain was sequenced by Songon. The Staphylococcus aureus strains revealed positive results of the presence of 292bp, 205bp, 524bp and 642bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus target gene teste The detection of Staphylococcus aureus which was isolated from dairy cow with mastitis showed that clfa A, clfa B, fnbp A and fnbp B were amplified in 26, 12, 28 and 3 isolates, respectively.