奶及奶制品中副结核分枝杆菌存活状态快速鉴定方法的建立
发布时间：2018-10-29 17:49
【摘要】：副结核病(Paratuberculosis)是由副结核分枝杆菌,即鸟分枝杆菌副结核亚种(Mycobacterium avium subsp.Paratuberculosis,Map)引起的一种以反刍动物为主的慢性消耗性传染病,也称Johne's病。该病很难净化,而且与人类的克罗恩氏(Crohn's)病存在潜在联系,因此,早期确诊感染动物,对防治本病具有重要意义。本实验为建立快速鉴定Map的检测方法,使用高特异性DNA探针技术,在制备的液体培养基中培养副结核分枝杆菌P10和P18两种菌株,提取基因组DNA,应用DNAStar软件将Genbank库中历年来发布的所有Map基因片段进行了比对,选出该菌ISMAV2基因的共有序列和特异性基因片段,按照引物和探针设计原则,应用Primer Express Software v2.0软件来设计引物和探针,成功构建重组质粒,用于建立TaqMan PCR标准曲线,通过特异性试验和敏感性试验,建立了一种可快速检测Map的TaqMan荧光定量PCR方法,实验结果表明该方法具有很高的特异性,且该检测方法的成功构建对奶及奶制品中Map的快速检测及早期诊断具有重要意义。通过应用所建立的TaqMan荧光定量PCR方法对不同批次的奶及奶制品样本进行了检测,并通过琼脂糖凝胶电泳进行了进一步的验证,结果发现44%的奶及奶制品样本检测结果均为Map阳性。因此为了建立一种快速鉴别诊断Map活／死菌的方法,来进一步的判定阳性结果中Map是否为活菌,本实验选取上述试验中同一样本不同批次判定结果为阴性的几种奶及奶制品样本,通过人工制备巴氏灭菌奶(把加有Map的奶样置于80℃灭活20min),将PMA染料与所建立的TaqMan荧光定量PCR方法结合,根据PMA只能够与死菌的DNA分子结合并抑制死菌扩增的原理,通过对PMA质量浓度和光照反应条件的优化,确定最佳反应条件,并在优化好的条件下,应用PMA对沸水浴灭活后奶样中的Map进行处理,通过TaqMan荧光定量PCR检测人工灭活奶样中Map的存活状态。结果显示：PMA在质量浓度为10μg/mL, BLU-V系统光照反应时间为20 min时,PMA能有效的与死菌DNA结合,且不影响活菌的扩增：TaqMan荧光定量PCR结果判定为Map阳性的奶样中未检测出活菌。
[Abstract]:Paratuberous (Paratuberculosis) is a chronic consumable infectious disease caused by Mycobacterium paratuberculosis (Mycobacterium avium subsp.Paratuberculosis,Map), also known as Johne's disease, which is mainly caused by ruminants. The disease is difficult to purify and has a potential link with human Crohn's (Crohn's) disease. Therefore, the early diagnosis of infected animals is of great significance in the prevention and treatment of this disease. In order to establish a method for rapid identification of Map, two strains of Mycobacterium paratuberculosis, P10 and P18, were cultured in liquid medium with high specificity DNA probe technique, and genomic DNA, was extracted. DNAStar software was used to compare all the Map gene fragments published in the Genbank library over the years. The common sequence and specific gene fragment of ISMAV2 gene were selected. The primers and probes were designed according to the principles of primer and probe design. Using Primer Express Software v2.0 software to design primers and probes, the recombinant plasmid was successfully constructed, which was used to establish the standard curve of TaqMan PCR. By using specificity test and sensitivity test, a TaqMan fluorescence quantitative PCR method was established for rapid detection of Map. The experimental results show that this method has high specificity, and the successful construction of this method is of great significance for the rapid detection and early diagnosis of Map in milk and dairy products. The samples of different batches of milk and dairy products were detected by TaqMan fluorescence quantitative PCR method, and further verified by agarose gel electrophoresis. The results showed that 44% of milk and milk samples were Map positive. Therefore, in order to establish a rapid method for differential diagnosis of Map live / dead bacteria, to further determine whether Map is a living bacterium in positive results. In this experiment, several kinds of milk and milk products which were negative in different batches of the same sample were selected, and the pasteurized milk was prepared manually (the milk sample with Map was placed at 80 鈩，
本文编号：2298368
[Abstract]:Paratuberous (Paratuberculosis) is a chronic consumable infectious disease caused by Mycobacterium paratuberculosis (Mycobacterium avium subsp.Paratuberculosis,Map), also known as Johne's disease, which is mainly caused by ruminants. The disease is difficult to purify and has a potential link with human Crohn's (Crohn's) disease. Therefore, the early diagnosis of infected animals is of great significance in the prevention and treatment of this disease. In order to establish a method for rapid identification of Map, two strains of Mycobacterium paratuberculosis, P10 and P18, were cultured in liquid medium with high specificity DNA probe technique, and genomic DNA, was extracted. DNAStar software was used to compare all the Map gene fragments published in the Genbank library over the years. The common sequence and specific gene fragment of ISMAV2 gene were selected. The primers and probes were designed according to the principles of primer and probe design. Using Primer Express Software v2.0 software to design primers and probes, the recombinant plasmid was successfully constructed, which was used to establish the standard curve of TaqMan PCR. By using specificity test and sensitivity test, a TaqMan fluorescence quantitative PCR method was established for rapid detection of Map. The experimental results show that this method has high specificity, and the successful construction of this method is of great significance for the rapid detection and early diagnosis of Map in milk and dairy products. The samples of different batches of milk and dairy products were detected by TaqMan fluorescence quantitative PCR method, and further verified by agarose gel electrophoresis. The results showed that 44% of milk and milk samples were Map positive. Therefore, in order to establish a rapid method for differential diagnosis of Map live / dead bacteria, to further determine whether Map is a living bacterium in positive results. In this experiment, several kinds of milk and milk products which were negative in different batches of the same sample were selected, and the pasteurized milk was prepared manually (the milk sample with Map was placed at 80 鈩，
本文编号：2298368
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