奶牛乳房炎无乳链球菌的分离及PCR鉴定

Isolation and PCR-identification of Streptococcus agalactiaee in milk sampled from mastitic dairy cows

利用THB(Todd-Hewitt Broth)固体培养基和色素试验培养基选择培养无乳链球菌,参照GenBank中无乳链球菌参考菌株(Accession:AF015927.1、JQ289582.1)16SrRNA和种属特异性基因cfb(CAMP因子)序列设计引物,对奶样中分离的12株疑似无乳链球菌进行鉴定。结果显示,经PCR扩增后,被检测的12株细菌均可扩增出预期大小的16S rRNA基因序列,而12株中有8株可以扩增出预期大小的cfb基因序列,条带单一,特异性好。序列BLAST显示,12株菌的16S rRNA基因序列与NCBI上报道的无乳链球菌相应序列高度同源(>99.0%),各分离菌株间的16S rRNA基因序列也高度同源(99.0%～100.0%);cfb基因序列与NCBI上已报道的无乳链球菌相应序列具有高度同源性(>99.0%),各菌株间cfb基因序列也高度同源(100.0%)。经选择培养与PCR鉴定结果可以确定12株疑似菌株中有8株为无乳链球菌。

THB selective solid medium and Islam medium were used to selectively culture the Streptococcus agalactiaee ,and primers of Streptococcus agalactiae 16S rRNA and its species-specificity gene cfb （CAMP factor）was designed according to the reference strain （Accession： AF015927.1,JQ289582.1） ,to identify 12 strains of suspected Streptococcus agalactiae. The results showed that 8 of the 12 suspected strains were Streptococcus agalactiaee according to their biochemical character and PCR identification. Online BLAST showed that 16S rRNA possessed high similarities among the 12 strains（99.0%-100.0%）and with their counterparts registered in GenBank（〉99.8%）. Similarly,the cfb gene is with high similarity among the 8 strains（100.0%） and with other Streptococcus agalactiaee logged-in NCBI（〉99.0%）. Therefore,according to selective cultivation and PCR characterization,8 strains were identified as Streptococcus agalactiaee.