应用双重PCR技术快速鉴定结核与非结核分枝杆菌

IDENTIFYING OF MYCOBACTERIUM TUBERCULOSIS AND NON-MYCOBACTERIUM TUBERCULOSIS BY DUPLEX PCR

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。

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 ouly amplify multi - mycobacterium tuberculosis strains, and beth 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/μl land 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.