猪葡萄球菌脱落毒素多重PCR检测方法的建立与初步应用  

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

作　　者：陈亚强[1,2] 郝永清[1] 张乐宜[2] 蔡汝健[2] 宋长绪[2] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]广东省农业科学院兽医研究所广东省兽医公共卫生公共实验室,广东广州510460

出　　处：《中国兽医学报》2012年第10期1452-1456,共5页Chinese Journal of Veterinary Science

基　　金：广东省生猪科技创新体系岗位专家专项(2008A020100020);广东省农业科技攻关项目(2007A020300006);广东省自然基金重点项目(06105311);促进科技服务业发展专项(2010A040301010)

摘　　要：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。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.

关 键 词：猪葡萄球菌 猪渗出性皮炎 表皮脱落毒素 多重PCR检测 

分 类 号：S852.61[农业科学—基础兽医学]