一种基于16SrRNA基因检测猪嗜血支原体PCR方法的建立  

Development of a PCR-based assay using the 16S rRNA gene for detection of Mycoplasma suis infection

作　　者：文辉强[1] 刘永刚[1] 石文达[1] 武嘉男[1] 姜成刚[1] 张交儿[1] 蔡雪辉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物疫病诊断及技术服务中心,黑龙江哈尔滨150001

出　　处：《中国兽医杂志》2013年第9期18-20,23,共4页Chinese Journal of Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(2012-ZL079)

摘　　要：本试验目的是建立一种基于16SrRNA基因检测猪嗜血支原体的PCR方法。根据GenBank上猪嗜血支原体16S rRNA基因(ID:U88565)设计特异引物,对哈尔滨采集病料进行PCR并克隆pMD18-T,测序所扩增的片段为411bp,同源性分析表明,该序列与参考序列同源性97.57%。特异性和敏感性试验表明,所建立的PCR诊断方法对猪肺炎支原体、大肠杆菌、沙门菌、葡萄球菌无交叉反应;血液基因组DNA最小检出量为0.75fg/μL。该方法具有快速、特异、敏感等特点,为猪嗜血支原体病的快速诊断及流行病学调查提供了新的手段。Our aim was to develop a PCR assay using 16S rRNA for detection of M. suis. The 16S rRNA of M. suis was amplified using primers developed from GenBank. The fragment of M. suis extrac- ted from clinical samples in Harbin was subsequently cloned into pMD18-T and sequenced. A 411 bp prod- uct was selectively amplified and phylogenetic analysis revealed that it was 97.57~ identical to GenBank accession U88565. No PCR products were amplified from the purified DNA of M. hyopneumoniae, E. co- li, S. typhimurium and S. aureus, while it was estimated that as few as 0.75 fg L-1 M. suis genomic DNA could be detected. The results showed that this PCR assay developed was an effective tool for diag- nosing M. suis infection in pigs with less time, high specificity and sensitivity. It is recommended for ap- plication in clinical diagnosis and epidemiologic survey of M. suis infection.

关 键 词：猪嗜血支原体 PCR 16S RRNA基因 

分 类 号：S852.7[农业科学—基础兽医学]