罗非鱼无乳链球菌分子血清型、耐药性以及快速检测方法的建立-水产养殖专业毕业论文.docxVIP

上海海 上海海洋大学硕士学位论文 II II GreenⅠ，通过观察其颜色变化判定结果。利用LAMP方法同时对无乳链球菌、海 豚链球菌、金黄色葡萄球菌、嗜水气单胞菌、温和气单胞菌、浅绿气球菌、变形 杆菌以及迟钝爱德华菌进行等温扩增，结果表明所建立的LAMP方法能够特异的检 测出无乳链球菌。对LAMP方法的灵敏度进行检测，发现其对纯DNA检测的最低 限度为10pg/反应，对纯细菌培养物的最低检测限度为20个/反应。LAMP检测方法 的灵敏度是普通PCR方法的10倍以上。表明以cfb作为靶基因所建立的LAMP检测方 法具有高度的特异性、敏感性以及稳定性，为罗非鱼无乳链球菌的快速检测提供 了新的技术。 3 罗非鱼无乳链球菌对喹诺酮类的耐药性及与其耐药基因的相关研究 为了解罗非鱼无乳链球菌体外对喹诺酮类药物耐药性及与其耐药相关基因 gyrA和parC基因突变的关系，本实验通过微量稀释法测定了41株分离菌株对4种喹 诺酮类药物的最小抑菌浓度（MIC）值，并克隆了相应菌株体内的gyrA和parC基因 测序分析各耐药基因突变情况。结果表明：41株罗非鱼无乳链球菌分离菌株对左 氧氟沙星、氟罗沙星、洛美沙星和依诺沙星等4种喹诺酮类抗菌药物的耐药率分别 为22.0%、75.6%、73.2%和100%。9株菌对4种喹诺酮类抗菌药物均表现耐药，其 中有6株菌发生了氨基酸突变。发生gyrA和parC基因突变的各有3株菌，另外3株菌 则未发现突变位点，这说明无乳链球菌对喹诺酮类药物耐药性的产生可能还存在 其他耐药机制。 关键词：罗非鱼，无乳链球菌，鉴定，分子血清型，环介导等温扩增快速检 测，最小抑菌浓度，耐药基因 PAGE PAGE IV Molecular Serotype，Drug-Resistance and Rapid Detection of Streptococcus agalactiae from Tilapia Abstract In 2009 disease outbreaks happened in tilapia farms in Southern China. High infectivity(50%) and high mortalities (95%) were observed in some farms. The disease constrained the tilapia aquaculture development. By biochemistry assays and molecular analysis of 16S rRNA, the pathogen was identified as Streptococcus agalactiae. That bacteria distributes widely in the world and can cause many disease in human, cattle and fish. This paper presented our reseach work on isolation and identification of S.agalactiae, their molecular serotyping, the establishment of loop-mediated isothermal amplification method for rapid detection, the minimal inhibitory concentration about quinolones and amplification of the resistance genes.The main results are as follows. Molecular serotype and drug-resistance of Streptococcus agalactiae from tilapia The goal of the present study is to investigate the relationship between serotype of Streptococcus agalactiae and its resistance to antibiotic. Twenty four bacteria strains were isolated from infected tilapia in several farms of Hainan province. With the Rapid ID32strep technique, morphological, regular physiological and biochemical a