利用RFLP分型方法鉴定牛源性链球菌和肠球菌  

Identification of Streptococcus spp. and Enterococcus spp. from mastitic dairy cows by restriction fragment length polymorphism analysis

作　　者：蔡金山[1] 阚威[2] 赵兴绪[1] 殷宏[3] 

机构地区：[1]甘肃农业大学动物医学学院,甘肃兰州730070 [2]青海省动物疫病预防控制中心,青海西宁810001 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《中国预防兽医学报》2016年第6期467-470,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十二五”农村领域国家科技计划(2011AA10A210);兰州市科技局科技三项(033143)

摘　　要：为分离及种属鉴定引起牛乳腺炎相关的链球菌和肠球菌,本研究于兰州及周边地区采集疑似奶牛乳腺炎乳样382份,通过THB(Todd-Hewitt Broth)固体选择培养基初步分离到67株疑似链球菌或疑似肠球菌。参照已发表文献合成链球菌属16S r RNA和16S^23S r RNA间隔区基因引物序列,扩增分离菌株16S^23S r RNA间隔区序列,产物分别利用AluⅠ和RsaⅠ单酶切消化,并以参考菌株的16S^23S r RNA酶切图谱为参考,对分离株进行限制性片段多态性(RFLP)分类分析;再选取各RFLP类群的任一菌株,扩增其16S r RNA基因并测序,并经NCBI核酸数据库进行比对,结果显示67株疑似链球菌中有53株为粪肠球菌(79.1%)、3株为屎肠球菌(4.5%)、3株为肠道肠球菌(4.5%)、8株为无乳链球菌(11.9%)。结果表明肠球菌属细菌(粪肠球菌、肠道肠球菌、屎肠球菌)与兰州市及周边地区奶牛乳腺炎的发病紧密相关,肠球菌属细菌与链球菌属细菌16S r RNA基因同源性很高,但可以通过分子生物学方法准确区分。To isolate and identify the Streptococcus spp. and Enterococcus spp. causing bovine mastitis, a total of 382 milk samples from dairy cows with suspected mastitis were collected and 67 isolates assumed as Streptococcus Enterococcus were primary isolated through THB （Todd-Hewitt Broth） selective medium. Primers of Streptococcus spp. 16S-23S rRNA were used for restriction fragment length polymorphism （RFLP） analysis by A1u I and Rsa I digestion for grouping the 67 isolates. Then, 16S rRNA genes of the isolates were sequenced to compare with the sequences of reference bacteria in GenBank. The results showed that 53 isolates （79.1%） were E.faecalis, 3 isolates （4.5%） were E.faecium, 3 isolates （4.5%） were E.hirae and 8 isolates （11.9%） were S.agalactiaee. These results revealed that the Enterococcus spp. was closely related with mastiffs within and around Gansu areas, and its 16S rRNA is of high homology with Streptococcus spp., but could be distinguished by sequencing analysis.

关 键 词：奶牛乳腺炎 链球菌 肠球菌 16S-23S rRNA RFLP 

分 类 号：S852.61[农业科学—基础兽医学]