猪葡萄球菌脱落毒素多重PCR检测方法的建立与初步应用

根据GenBank已发表的猪葡萄球菌的6种脱落毒素基因序列,设计合成了6对相应的特异性引物,通过特异性、敏感性和重复性试验建立了可行的多重PCR检测方法。用该方法对临床分离到的9株猪葡萄球菌进行检测,均扩增出了与预期大小相符的23SrDNA（662bp）条带;同时,其中6株分别扩增出了EXHA（316bp,2株）、EXHC（525bp,2株）和Shet-A（814bp,2株）基因特异性条带;另外3株均未扩增出任何毒素基因特异性条带,鉴定为无毒力菌株,以上结果与生化鉴定及单一PCR检测测序结果一致。结果表明,本试验所建立的多重PCR方法不仅操作快速方便、节约试验成本,而且具有高度特异性、敏感性和良好的重复性,可用于仔猪渗出性皮炎的诊断和猪葡萄球菌的快速鉴定。

Establishment and preliminary application of a multiplex PCR assay for detection of exfoliative toxin of Staphylococcus hyicus

In this study six pairs of primers were designed and synthesized according to the reported sequences of six different exfoliative toxin types of Staphylococcus hyicus in GenBank.A multiplex PCR assay was established after specific,sensitive and accuracy tests.Nine clinical isolates of Staphylococcus hyicus were detected by this multiplex PCR assay,23S rDNA（size of 662 bp） were amplified from all of the nine strains as expected.At the same time specific fragments targeted EXHA（size of 316 bp,2 strains）,EXHC（size of 525 bp,2 strains）,Shet-A（size of 814 bp,2 strains） genes were amplified from six isolates,respectively.None of the specific fragments of toxin genes were amplified from the other three isolates,indicating no virulence of these three isolates.The above results were correspondent to the results of biochemical identification and single PCR detection and sequencing.The results showed that this new method was not only rapid and convenient,but also highly specific,sensitive and repeatable.It can be used for detection of exudative epidermitis and the fast identification of Staphylococcus hyicus.