9型猪链球菌广东株的分离鉴定和基因序列分析

Isolation,Identification and Gene Sequence Analysis of Streptococcus suis Serotype 9 Strain from Guangdong

为了解9型猪链球菌广东分离株毒力因子基因型、基因变异和进化情况,本试验采集发病猪脑脊液、关节液、脾脏、肝脏、淋巴结进行细菌分离培养和生化鉴定,采用PCR方法鉴定其血清型及部分毒力基因,对cps9D、gdh、gapdh与orf2基因进行序列测定和分析,并构建系统发育树。结果显示,分离菌镜检为革兰氏阳性链状球菌,在鲜血琼脂平板中呈β溶血,可发酵大多数糖类,能成功扩增cps9D基因,含重要的保守基因gdh及毒力基因gapdh和orf2,表明分离菌株为9型猪链球菌。分离菌株cps9D、gdh、gapdh、orf2基因核苷酸序列与GenBank上国内外参考菌株的同源性分别为95.9%~100.0%、98.6%~99.8%、98.7%~99.6%和95.5%~99.9%,说明分离菌株与国内外其他9型猪链球菌毒株核苷酸同源性都较高,且与国内外的流行毒株基本一致,未发生太大的变异。系统发育树表明,分离株与国内外来源不同的猪链球菌分离株同源性高,亲缘关系密切。

In order to understand the virulence factor,gene mutation and evolution of Streptococcus suis serotype 9 strain from Guangdong,the cerebrospinal fluid,joint fluid,spleen,liver,lung and lymph node were collected for bacterium culture and biochemical identification,and PCR method was used to identify its serotype and the partial virulence genes.cps9D,gdh,gapdh and orf 2 genes were sequenced and analyzed,and phylogenetic trees were constructed.The results showed that the isolate was gram-positive chain cocci,β-hemolysis in blood agar plate,and could ferment most sugars,and amplify cps9D gene successfully,including important conservative gene gdh and virulence genes gapdh and orf 2,which proved that it was Streptococcus suis serotype 9.The nucleotide homologies of cps9D,gdh,gapdh and orf 2 genes of the isolate with different strains at home and abroad in GenBank were 95.9%to 100.0%,98.6%to 99.8%,98.7%to 99.6%and 95.5%to 99.9%,respectively.It showed that the isolate had higher nucleotide homology with other Streptococcus suis serotype 9 strains at home and abroad,which was basically consistent with the epidemic strains at home and abroad,and there was not much variation.Phylogenetic tree showed that the isolate had high homology and close relationship with Streptococcus suis isolates from different sources at home and abroad,and they were closely related.