论文部分内容阅读
为了建立快速、敏感的聚合酶链式反应(PCR)诊断斑点叉尾鮰Ictalurus punctatus的肠败血症技术,作者根据NCB I公布的鮰爱德华氏细菌序列,设计了一对特异性诊断引物CM3/CM4,对鮰爱德华氏细菌特异基因片段进行PCR扩增试验,PCR反应条件的优化以及不同检测材料的比较,同时测试了该方法的特异性和敏感性,并对广西4个市县临床样品进行了检测。结果表明:用该引物可以扩增到与预计大小相符的276 bp鮰爱德华氏菌特异性片段;PCR反应与Ⅰ型荧光假单胞菌、点状产气单胞菌点状亚种、鳗弧菌、温和气单胞菌、肠型点状产气单胞菌、柱状黄杆菌、嗜水气单胞菌、河弧菌、迟缓爱德华氏菌及海豚链球菌无交叉反应;用该PCR法可以检测出病鱼脑、肝、肾及脾中的病原菌,检测的最低细菌数为12个,且病鱼内脏组织、菌落及菌液均可直接用于该PCR扩增;临床菌株检测结果与基于菌株16S rRNA基因序列系统进化分析和生化鉴定结果一致。因此,本试验中所建立的PCR检测方法在斑点叉尾鮰肠败血症的诊断和防治方面具有较好的应用前景。
In order to establish a rapid and sensitive polymerase chain reaction (PCR) for the diagnosis of enterocolitis in the catfish Ictalurus punctatus, a pair of specific diagnostic primers CM3 / CM4 was designed according to the sequence of E. bovis published by NCB I. PCR amplification of Edwardsiella bacteria-specific gene fragments, optimization of PCR reaction conditions and comparison of different test materials were performed. The specificity and sensitivity of this method were also tested. The clinical samples of 4 municipalities and counties in Guangxi were also tested. The results showed that this primer could amplify the 276 bp of the specific fragment of Edwardsiella in accordance with the expected size. The PCR reaction was similar to that of Pseudomonas fluorescens Ⅰ, Pseudomonas punctatus, Bacillus subtilis, Aeromonas sobria, Aeromonas punctatus, Flavobacterium rodigum, Aeromonas hydrophila, Vibrio cholerae, Edwardsiella tarda and Streptococcus pulexia. By this PCR method, Detection of pathogens in the brain, liver, kidney and spleen pathogens, the detection of the minimum number of bacteria is 12, and diseased fish visceral tissue, colonies and bacteria can be used directly for the PCR amplification; clinical strains and test results based on The phylogenetic analysis of the 16S rRNA gene of the strain was consistent with the biochemical identification results. Therefore, the PCR detection method established in this experiment has a good application prospect in the diagnosis and prevention of catfish intestinal cats.