气肿疽梭菌PCR检测方法的建立及初步应用

发布时间：2018-04-11 02:25

本文选题：聚合酶链式反应 + 气肿疽梭菌　；参考：《延边大学》2015年硕士论文

【摘要】：气肿疽是由气肿疽梭菌引起的以牛为主的一种热性、急性、地方性传染病,以肌肉和深部组织发生气性坏疽为主要特征。该病主要经过皮肤及消化道创伤感染,往往来不及治疗就会导致病畜死亡,羊、猪、鹿甚至是人也都有相关报道显示被感染,被国际兽医局评定为第三类传染病。气肿疽在0.5～3岁的黄牛及水牛多发,无明显的季节规律。气肿疽在我国的暴发,给我国的畜牧业带来了巨大的经济损失,也引起了人们的高度关注。但目前尚无有效的防控手段,因此,建立快速、敏感且特异的检测方法来诊断气肿疽病是当务之急。本试验为建立气肿疽梭菌(Clostridium chauvoei)的快速检测方法,根据GenBank中已发表的气肿疽梭菌细胞毒素cctA基因序列(登录号：JQ692583)设计了一对特异性引物,并以气肿疽梭菌标准株C54-1全基因组DNA为模板,对气肿疽梭菌cctA基因进行PCR扩增,并经过敏感性和特异性试验对该试验方法进行验证。结果显示：建立的气肿疽梭菌PCR检测方法扩增出的片段大小为501bp,与GenBank上气肿疽梭菌的同源性达99.4%,且该方法与D型产气荚膜梭菌、E型产气荚膜梭菌、腐败梭菌和巴氏杆菌等病原体无交叉反应,最低检测浓度为12.30 fg/μL。结果表明,建立的PCR检测方法具有快速、特异、敏感、高效等优点,与其他诊断方法相比更适用于气肿疽梭菌的诊断,为牛气肿疽病的快速检测和诊断提供基础研究。
[Abstract]:Emphysema is a hot, acute, endemic infectious disease caused by Clostridium emphysema. It is characterized by the occurrence of gaseous gangrene in muscles and deep tissues.The disease mainly passes through the skin and the digestive tract wound infection, often causes the disease animal death without the time treatment, sheep, the pig, the deer and even the human also has the related report to show to be infected, is rated as the third kind infectious disease by the international veterinary bureau.Emphysema was more common in 3-year-old yellow cattle and buffalo with no obvious seasonal regularity.The outbreak of emphysema in our country has brought huge economic loss to animal husbandry in our country.However, there are no effective methods to prevent and control emphysema, so it is urgent to establish a rapid, sensitive and specific detection method for the diagnosis of emphysema.In order to establish a rapid detection method for Clostridium chauvoeii, a pair of specific primers were designed according to the cctA gene sequence of Clostridium chauvoeii published in GenBank (accession number: JQ692583).The whole genome DNA of C. emphysema strain C54-1 was used as a template to amplify the cctA gene of Clostridium emphysema by PCR, and the method was verified by sensitivity and specificity test.The results showed that the amplified fragment size of the established PCR detection method was 501bp, and the homology was 99.4 with that of Clostridium emphysema on GenBank, and the method was similar to that of Clostridium perfringens type D (Clostridium perfringens).There was no cross reaction between Clostridium putrefaciens and Pasteurella, and the lowest detection concentration was 12.30 fg/ 渭 L.The results showed that the established PCR detection method was rapid, specific, sensitive and efficient. Compared with other diagnostic methods, it was more suitable for the diagnosis of Clostridium emphysema, and provided the basic research for rapid detection and diagnosis of bovine emphysema.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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