奶及奶制品中副結(jié)核分枝桿菌存活狀態(tài)快速鑒定方法的建立
發(fā)布時間：2018-10-29 17:49
【摘要】：副結(jié)核病(Paratuberculosis)是由副結(jié)核分枝桿菌,即鳥分枝桿菌副結(jié)核亞種(Mycobacterium avium subsp.Paratuberculosis,Map)引起的一種以反芻動物為主的慢性消耗性傳染病,也稱Johne's病。該病很難凈化,而且與人類的克羅恩氏(Crohn's)病存在潛在聯(lián)系,因此,早期確診感染動物,對防治本病具有重要意義。本實驗為建立快速鑒定Map的檢測方法,使用高特異性DNA探針技術(shù),在制備的液體培養(yǎng)基中培養(yǎng)副結(jié)核分枝桿菌P10和P18兩種菌株,提取基因組DNA,應(yīng)用DNAStar軟件將Genbank庫中歷年來發(fā)布的所有Map基因片段進行了比對,選出該菌ISMAV2基因的共有序列和特異性基因片段,按照引物和探針設(shè)計原則,應(yīng)用Primer Express Software v2.0軟件來設(shè)計引物和探針,成功構(gòu)建重組質(zhì)粒,用于建立TaqMan PCR標準曲線,通過特異性試驗和敏感性試驗,建立了一種可快速檢測Map的TaqMan熒光定量PCR方法,實驗結(jié)果表明該方法具有很高的特異性,且該檢測方法的成功構(gòu)建對奶及奶制品中Map的快速檢測及早期診斷具有重要意義。通過應(yīng)用所建立的TaqMan熒光定量PCR方法對不同批次的奶及奶制品樣本進行了檢測,并通過瓊脂糖凝膠電泳進行了進一步的驗證,結(jié)果發(fā)現(xiàn)44%的奶及奶制品樣本檢測結(jié)果均為Map陽性。因此為了建立一種快速鑒別診斷Map活／死菌的方法,來進一步的判定陽性結(jié)果中Map是否為活菌,本實驗選取上述試驗中同一樣本不同批次判定結(jié)果為陰性的幾種奶及奶制品樣本,通過人工制備巴氏滅菌奶(把加有Map的奶樣置于80℃滅活20min),將PMA染料與所建立的TaqMan熒光定量PCR方法結(jié)合,根據(jù)PMA只能夠與死菌的DNA分子結(jié)合并抑制死菌擴增的原理,通過對PMA質(zhì)量濃度和光照反應(yīng)條件的優(yōu)化,確定最佳反應(yīng)條件,并在優(yōu)化好的條件下,應(yīng)用PMA對沸水浴滅活后奶樣中的Map進行處理,通過TaqMan熒光定量PCR檢測人工滅活奶樣中Map的存活狀態(tài)。結(jié)果顯示：PMA在質(zhì)量濃度為10μg/mL, BLU-V系統(tǒng)光照反應(yīng)時間為20 min時,PMA能有效的與死菌DNA結(jié)合,且不影響活菌的擴增：TaqMan熒光定量PCR結(jié)果判定為Map陽性的奶樣中未檢測出活菌。
[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，
本文編號：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，
本文編號：2298368
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