猪源粪肠球菌和屎肠球菌多重PCR快速鉴定方法的建立  

Development of a multiplex PCR assay for detection of swine-originated E.faecalis and E.faecium isolates

作　　者：王亚宾[1] 陈丽颖[1] 胡慧[1] 段志刚[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院兽医微生物实验室,河南郑州450002

出　　处：《中国兽医学报》2011年第8期1123-1127,共5页Chinese Journal of Veterinary Science

摘　　要：粪肠球菌和屎肠球菌是引起猪感染发病的优势肠球菌种,以肠球菌的16S rRNA基因设计属特异性引物,利用SodA基因多态性设计种特异性引物,同时优化反应条件,建立了能同时测定猪源粪肠球菌和屎肠球菌多重PCR方法。通过特异性和敏感性分析,该方法能扩增出肠球菌属特异性片段及粪肠球菌和屎肠球菌种特异性片段;对粪肠球菌敏感性为0.1ng DNA,屎肠球菌为1ng DNA。通过对来源于猪的临床菌株、粪便菌株和鲜猪肉菌株的肠球菌进行测定,均能成功扩增出属特异性片段和种特异性片段。经过与16SrRNA测序方法和快速生化鉴定试剂盒(Vitek-32)比较,多重PCR与16SrRNA测序方法对猪的临床菌株、粪便菌株和鲜猪肉菌株的鉴定符合率100%;与Vitek-32鉴定符合率为62.3%,其中,与分离于感染猪的临床菌株符合率仅有46.7%,特别是感染猪的屎肠球菌,符合率仅为22.3%。A method of multiplex PCR assay was developed for the quick detection of swine-originated Enterococcus faecalis and E.faecium isolates by using genus-primers based on the conserved regions 16S rRNA gene and species-specific primers on the SodA gene polymorphism.The specificity and sensitivity analysis showed that only Enterococci genus-and species-specific for E.faecalis and E.faecium fragments were obtained,with a minimal detectable dosage of 0.1 ng DNA for E.faecalis and that of 1 ng DNA for E.faecium.Genus-and species-fragments was both successfully amplified from the isolates of clinical,fecal and raw flesh origins.By comparison,the multiplex PCR results showed a 100% coincidence rate to those identified by the 16S rRNA sequencing;whilst the coincidence rate between the PCR and Vitec-32 identification results was only 62.3%,the clinical isolate showing a coincidence rate of 46.7% and it was especially low among the E.faecium isolates（22.3%）.

关 键 词：猪 肠球菌 多重PCR 16S RRNA VITEK-32 鉴定 

分 类 号：S852.6[农业科学—基础兽医学]