单因子和正交设计法建立分枝杆菌多重PCR体系  

Establishment of Mycobacterium Multiplex PCR System with Single Factor and Orthogonal Design Method

作　　者：周方永[1] 李岩 薄新文[3] 马勋[1] 

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]新疆兵团畜牧兽医工作总站,乌鲁木齐830000 [3]新疆农垦科学院,石河子832000

出　　处：《石河子大学学报（自然科学版）》2013年第4期449-456,共8页Journal of Shihezi University(Natural Science)

基　　金：国家科技支撑计划项目(2012BAD43B02)

摘　　要：为了建立一种快速区分鉴定结核与非结核分枝杆菌的多重PCR方法,以分支杆菌属特异基因32kD,结核分枝杆菌种特异基因MTP40和结核分枝杆菌复合群特异基因IS6110为目标基因,设计合成3对特异性引物,以结核分枝杆菌标准株DNA为模板,通过对PCR反应体系中DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度和TaqDNA聚合酶浓度5个因素的正交设计,以及PCR反应条件中各反应参数的优化,建立了鉴别分支杆菌的多重PCR检测方法,并对该方法的特异性、敏感性进行了验证。结果显示:该方法对结核分枝杆菌标准株H37Ra能扩增出506、396和984bp,对卡介苗(BCG)能扩增出506和984bp片段,对禽分支杆菌、胞内分支杆菌、偶发分支杆菌、耻垢分支杆菌、龟分支杆菌能够扩增出506bp片段,对大肠杆菌等的扩增结果均为阴性。建立的多重PCR敏感度最低可检出6.6×10-6 ng。结论:该方法具有特异性高、敏感性好等特点,可用于奶牛结核分枝杆菌和非结核分枝杆菌的鉴别诊断。To establish a multiplex PCR system for rapid identification of Tuberculosis and Nontuberculous Mycobaeteria. This paper took Genus specific genes 32KD of Myeobacterium, species specific gene MTP40 of Mycobaeterium Tuberculosis and specific genes IS6110 as target genes of designed and synthesized three pairs of specific primers. With the standard strain of Myco-bacterium Tuberculosis DNA as a template,we constructed multiplex PCR system for identifying Myeobacterium through orthogonal the designs of five factors including DNA template concentration in the PCR reaction system, Mg2 ＋ concentration, dNTPs concentration,primers concentration and Taq DNA polymerase concentration,and through the optimization of various reaction parameters. The specificity and sensitivity of this system were validated. Results.. 506bp, 396bp and 984bp fragments were amplified in the standard strain of Mycobaeterium Tuberculosis H37Ra. 506bp and 984bp fragments were amplified in Guerin（BCG）. The 506bp fragment was amplified in Myeobacterium avian, Mycobacterium intracellulare, Myeobacterium fortuiturn, Mycobaeterium smegmatis and Mycobacterium chelona. The result of Escheriehia coli was negative. The sensitivity minimum of multiplex PCR was 6.6×10-8 ng. Conclusion：The method has a high specificity and sensitivity,and can be used in diagnostieation of Mycobacterium Tuberculosis and Nontuberculous Mycobacteria for cows.

关 键 词：分枝杆菌 多重PCR 假阳性 正交设计 

分 类 号：S852.61[农业科学—基础兽医学] Q78[农业科学—兽医学]