斑点叉尾鮰肠败血症PCR诊断方法的建立

Development of a diagnostic PCR assay for enteric septicemia in channel catfish Ictalurus punctatus

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。

Edwardsiella ictaluri as a causative pathogen of enteric septicemia of channel catfish（ Ictalurus punctatus） （ESC） is the most important disease affecting the channel catfish aquaculture industry. Good management practices including rapid and sensitive detection of the pathogen are necessary for effective control of the disease. We designed primers CM3/CM4 based on the sequences of E. ictaluri published in the NCBI to amplify the specific DNA fragment of E. ictaluri. The PCR reaction was optimized and clinical samples from 4 places of Guangxi were detected. Besides, the specific test, sensitive test and comparison of different materials for PCR amplification were also carried out. The results indicated that the CM3/CM4 primers set amplified a specific 276 bp DNA fragment from the pathogen, but not from the common bacteria in fisheries, Pseudomonas fluorescent biotype Ⅰ , Aeromonas punctata sub. , Vibrio anguillarum, Aeromonas sobria, Aeromonas punctata f. intertinalis, Flavobacterium columnare, Aeromonas hydrophila, Vibrio fluvialis, EdwardsieUa tarde and Streptococcus iniae. The minimal bacterial number the PCR can detect was below as few as 12 cells of Edwardsiella ictaluri. The PCR was also effective in detecting the bacterium from inoculated tissue of brain, liver, kidney and spleen. Furthermore, the materials including lawn and liquid culture were directly amplified in this PCR. The results of clinical strains by PCR were consistent with the phylogenetic analysis results base on 16S rRNA gene sequence and the biochemistry.