【作者】 孙见；

【导师】 武华；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2012， 硕士

【摘要】 水貂出血性肺炎是由铜绿假单胞菌感染引起的一种以发病水貂呼吸困难、口鼻流血为主要特征的烈性传染病，发病急、死亡快、呈地方性流行，已成为危害我国水貂养殖业最严重的细菌性传染病之一，给广大水貂养殖户造成了巨大的经济损失，因此开展对此病的研究迫在眉睫。铜绿假单胞菌曾称绿脓杆菌，是一种人兽共患性病原菌，属于革兰氏阴性短杆菌，有鞭毛，能单向运动。该菌对外界环境的抵抗力较强，在污染的环境及土壤中可长时间存活，对很多化学消毒剂和抗生素有抵抗力。本实验室从威海送检的发病水貂肺脏中分离到一株细菌，通过培养特性、生化试验和PCR方法鉴定为铜绿假单胞菌，命名为PA-WH-0808。致病性试验结果表明，8.0×1010CFU/mL的菌液10倍系列稀释后，感染60日龄健康昆明系小鼠，半数致死量和最小致死量分别为1.4×106.0CFU和8.0×105.0CFU。死亡小鼠剖检可见皮下和肺脏出血,并能从死亡小鼠的肺脏分离出与感染菌株形态特征完全一致的菌落。药敏试验结果显示，该分离菌株对庆大霉素、环丙沙星、诺氟沙星等较为敏感，而对青霉素、链霉素不敏感。系统发育分析结果表明，该分离菌株与从小麦中分离出的铜绿假单胞菌的亲缘关系较近，因此猜测该发病水貂可能是食用了污染的饲料引起的。我们采用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)，建立了一种可视化的快速检测铜绿假单胞菌的实验方法。该方法针对铜绿假单胞菌16SrDNA基因的保守区域设计了4条引物，在Bst DNA聚合酶的作用下，可以实现DNA的梯状等温扩增。优化LAMP的反应体系，并检验其灵敏性、特异性。PA-LAMP检测方法从核酸抽提到检测结果出现仅需60min，该方法具有良好的特异性，与感染毛皮动物的常见菌如大肠杆菌、沙门氏菌等无交叉反应，灵敏度是常规PCR的100倍。试验结果证明，PA-LAMP方法可快速、灵敏、特异地检测铜绿假单胞菌，在实验室和现场应用都具有良好的应用前景。更多还原

【Abstract】 Hemorrhagic pneumonia of mink is an acute infectious disease caused byPseudo mo na s a e rugin o sa, clinical signs are various for bleeding from the nose ormouth, dyspnoea and sudden death.This infectious disease makes the mosteconomic loss for the farmed mink in China, therefore it is necessary to carry outthis study of the disease.P se ud o mo n a s a erugino sa is a common bacterium that can cause disease inanimals and humans. It is a Gram-negative, rod-shaped bacterium with unipolarmotility by monotrichate flagellum. Pseudomonas aeruginosa has strong resistanceto the external environment, and can survive in a polluted environment and soil fora long time, and is resistant to many chemical disinfectants and antibiotics.A Pseudomonas aeruginosa was isolated from the lung of a dead mink fromWeihai of Shan Dong Province.We identified the Pseudomonas aeruginosa basedon colony characteristics,biochemical tests and PCR. Pathogenicity test showedthat the LD50for mice is1.4×106.0CFU,and the MLD is8.0×105.0CFU.Wesuccessfully isolated the same bacteria as infectious bacteria from the infectiousmice.Drug susceptibility test showed that the isolated was sensitive to some drugssuch as gentamicin,Ciproflaxin and norfloxacin,but resistant to penicillin andstreptomycin. On the basis of the bacterial16srRNA gene sequence phylogeneticanalysis and comparison of this gene sequence with sequence in RNA sequencedatabase, it was considered that the isolated was closely related to me mbers of thePseudo mo na s aerugino sa from the wheat,so we suepected that the isolated wasfrom the contaminated feed.LAMP is a rapid and visible method to detect the Pseudomonas aeruginosa.Aset of four primers were designed based on the PA-16SrDNA gene sequence byPrimerExplorerV4software. The specificity and sensitivity are very good byoptimizing reaction system and reaction time.It was showed that the Pseudomonsaaeru ginoa s could be detected within60minutes by this LAMP, and the sensitivitywas100times higher than that of general PCR. Establishment of LAMP provides anew alternative method for the rapid detection of Pseudomonsa aeruginoas,and hasa good prospect in the labortary and clinical tests.更多还原