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

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.