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为了从分子水平上对1株致病性罗非鱼链球菌进行分类学鉴定,利用原核生物16SrRNA基因通用引物对分离纯化的罗非鱼致病性链球菌进行16S rRNA基因的克隆及序列分析。结果扩增出长约1.5 kp目的片段,测序得到1条长度为1 447 bp核苷酸序列。核苷酸相似性分析表明,序列与NCB I公布的海豚链球菌(Streptococcus iniae,S.iniae)SCCF5L菌株16SrRNA基因核苷酸序列相似性最高(99.4%),暂称为中国广西株(S.iniae-CGX)。同时,亲源关系较近的S.iniae、S.difficilis和S.agalactiae代表菌株构建的系统发育进化树显示,所得菌株与S.iniae代表菌株组成同一进化分支,与S.agalactiae代表菌株组成的另一进化分支距离较近(95.5%),而与S.difficilis代表菌株组成的进化分支距离较远(92.3%)。上述研究证实,本试验从发病罗非鱼脑组织分离到的致病性链球菌为海豚链球菌。
In order to classify a virulent strain of pathogenic Streptococcus tilapia at the molecular level, 16S rRNA gene was cloned and sequenced by using 16S rRNA gene universal primers of prokaryotes to isolate and purify Streptococcus tilapia. As a result, a fragment of about 1.5 kp was amplified and one nucleotide sequence of 1 447 bp was obtained. Nucleotide similarity analysis showed that the nucleotide sequence of the 16S rRNA gene from Streptococcus iniae (SC) inoculated with NCB I was the highest (99.4%), and tentatively named as Guangxi strain. iniae-CGX). At the same time, the phylogenetic tree constructed by S.iniae, S.difficilis and S.agalactiae, which are closely related to each other, showed that the obtained strain and S.iniae represent the same evolutionary branch, which is composed of the S. alactiae strains The other branch of evolution was closer (95.5%), while the divergent branch from S. difficilis was 92.3%. The above study confirmed that the test from the incidence of tilapia brain tissue pathogenic Streptococcus is Streptococcus dolphin.