【摘要】 本研究建立1种双重PCR技术,用于区分鉴定结核与非结核分枝杆菌。在最佳扩增反应条件下,检测引物P1、P2和P3、P4扩增结核分枝杆菌的特异性及敏感性,并对19株结核分枝杆菌和9株非结核分枝杆菌临床分离株进行扩增鉴定。结果表明,引物P1、P2能扩增所试13株结核和非结核分枝杆菌,P3、P4仅扩增结核分枝杆菌复合群菌种,两对引物同时扩增3种非分枝杆菌均为阴性。两对引物双重PCR检测结核分枝杆菌DNA的敏感性分别为100pg/μl和10pg/μl。检测28株临床分离株,结核分枝杆菌可扩增出368bp和225bp2条带,非结核分枝杆菌仅扩增出一条361bp～383bp带。因此,该双重PCR技术的建立为结核及非结核分枝杆菌的快速鉴定提供了1个可供选择的手段。更多还原

【Abstract】 Identify mycobacterium tuberculosis and non-mycobacterium tuberculosis by double PCR technique.Objective:to establish double PCR technique to identify mycobacterium tuberculosis and non-mycobacterium tuberculosis.Approach:to select the best double PCR reacting condition,detect the specificity and the sensitivity of primer P1,P2 and primer P3,P4 in mycobacterium amplification,and identify 28 CLIN separating mycobacterium tuberculosis and non-mycobacterium tuberculosis strains through experiment.Result:primer P1,P2 can amplify 18 sample mycobacterium tuberculosis strains while primer P3,P4 can only amplify multi-mycobacterium tuberculosis strains,and both primer prove negative in amplification of the 4 non-mycobacterium tuberculosis strains.The result shows that amplifying the 16SrRNA 361bp-383bp gene fragment of Mycobacterium tuberculosis by primer P1,P2 and amplifying the 16SrRNA 225bp gene fragment of multi-mycobacterium tuberculosis by primer P3,P4 are specific in mycobacterium tuberculosis and non-mycobacterium tuberculosis identification.The detection sensitivity of the primer P1,P2 and P3,P4 are 100pg/μ1and 10pg/μl.In double PCR detection of 28 sample CLIN separating strains,368bp and 225bp gene fragments amplification bands are seen in mycobacterium tuberculosis while only one 361bp-383bp gene fragment amplification band is seen in non-mycobacterium tuberculosis.According to the above difference,mycobacterium tuberculosis and non-mycobacterium tuberculosis can be identified by experiment within 5 hours.Conclusion:double PCR technique provides an option to make a fast identification of mycobacterium tuberculosis and non-mycobacterium tuberculosis.更多还原