广西罗非鱼无乳链球菌病病原分离、鉴定及毒力基因检测分析-水产养殖专业论文.docxVIP

II II 在 80%以上。 采用 PCR 方法对 30 株无乳链球菌的 cfb、cpsE 和 sip 3 种毒力基因进 行检测，结果表明，30 株菌株为同一基因型 cfb+cpsE+sip+。在 30 株无乳链 球菌中，根据来源地，选取具有代表性的 6 株对其 cfb、cpsE 和 sip 基因进 行了克隆和测序分析，结果显示，无乳链球菌广西分离株 cfb、cpsE、sip 基因核苷酸的同源性分别在 99.1%～100%、99.6%～100%和 99.5%～100%， 氨基酸的同源性分别在 98.8%～99.6%、100%、99.1%～100%之间。基于广 西分离株的 3 个毒力基因遗传进化树分析，无乳链球菌广西分离株的 3 个 毒力基因各聚在同一个群内，与国内各基因相应参考菌株 JQ289564（广东）、 HQ148672（广东）、HQ148671（广东）、JQ289567（海南）、HQ888682 （海南）、EF694027（山东）同源性高达 97.7%～100%，表明无乳链球菌 广西分离株亲缘关系很近，与国内参考菌株亲缘关系也较近。 关键词：罗非鱼 无乳链球菌 毒力基因型 克隆 III III ISOLATION, IDENTIFICATION AND DETECTION, ANALYSIS OF VIRULENCE GENE OF TILAPIA IN GUANGXI WITH STREPTOCOCCUS AGALACTIAE DISEASE ABSTRACT In recent years, Stretococcus agalactiae has become an important pathogenic bacterium of tilapia’s bacteriosis, which causes tilapia industry great economic losses. Through investigation, it is found that Streptococcus iniae and Streptoccus agalactiae are the main reasons for tilapia’s streptococcicosis recently. Thirty Stretococcus agalactiae strains (Nanning 7, Qinzhou City 3, Beihai 7, Liuzhou 5, Wuzhou City 1, Yulin City 7) are isolated from 672 moribund tilapias witch collected in 12 counties and 8 cities of Guang Xi province, the rate is 4.46%. And these isolates are identified by API 20 Srep and 16S rRNA molecular biological characterization. Also there are 9 strains of which are not hemolytic, the remaining 21 strains of test hemolysis are positive. Analysising the similarities of the 30 test strains， 16S rRNA gene sequences and counterparts of streptococcus registered in GenBank with BLAST, showed that 16S rRNA of all the strains possessed high similarities with Streptococcus agalactiae (JF423947 isolated from Hainan province, JQ039375 and KF111315 isolated from Guangdong province, JQ990156 isolated from Guangxi province) (98.5％～100％), possessed relatively low similarities with streptococcus iniae (EU622514/FJ870987) (93.4% ～ 95.8%), streptococcus dysgalactiae (AB159678/X59030) (88.9% ～ 96.4%) and Streptococcus pyogenes (AB