关键词: 结核分枝杆菌, 抗药性, 细菌, 微生物敏感性试验, 寡核苷酸序列分析

Abstract: Objective Study on gene chip technique in detection of drug resistance of Mycobacterium tuberculosis and its clinical value.  Methods (1) One hundred and thirty-seven strain identification of Mycobacterium clinical isolates from Changsha Central Hospital by gene chip and Spoligotyping technology from 2011-01 to 2012-12. (2)Drug-susceptibility test of rifampin and isoniazid to Mycobacterium tuberculosis clinical isolates by absolute concentration method. (3) Drug-susceptibility test of rifampin and isoniazid to Mycobacterium tuberculosis clinical isolates by DNA chips for rpoB, KatG, inhA. Rifampin resistant is one or a plurality of site mutation of rpoB gene, isoniazid resistance is one or a plurality of site mutation of KatG gene and inhA gene. Compared Spoligotyping with gene chip method of species identification by Kappa test, Compared the absolute concentration method with gene chip method of drug sensitivity by χ² test and Kappa test.  Results （1）For 45 rifampin sensitive (under 50 μg/ml) and isoniazid sensitive(under 1 μg/ml) isolates tested by absolute concentration method, 42 are wild type for rpoB gene with the sensitivity of 93.3%(1 for 511T-C mutations, 1 for 531C-T mutations, 1 for 516A-T mutations), 45 are wild type for KatG gene with the sensitivity of 100.0% and 43 are wild type for inhA gene with the sensitivity of 95.6%(2 for15C-T mutation) by DNA chip method, respectively. （2）For 18 rifampin-resistant (50－250 μg/ml) isolates tested by absolute concentration method, 16 has mutation in rpoB gene with the sensitivity of 88.9% by DNA chip method. Those mutations happened at 531, 516, 526, 511 amino acid sites of rpoB. For 19 rifampin-resistant (above 250 μg/ml) isolates tested by absolute concentration method, 19 has mutation in rpoB gene with the sensitivity of 100% by DNA chip method. Those mutations happened at 531, 516, 526, 511 amino acid sites of rpoB.（3）Compare the DNA chips with the absolute concentration method, for 13 isoniazid-resistance(1－10 μg/ml) isolates, 12 has mutation in KatG gene with the sensitivity of 92.3% by DNA chip method, which mainly happened at 315 site. No mutation related to the drug resistance was found in inhA gene. Compare the DNA chips with the absolute Concentration method, For 9 isoniazid-resistance(above 10 μg/ml) isolates, 9 has mutation in KatG gene with the sensitivity of 100% by DNA chip method, which mainly happened at 315 site. No mutation related to the drug resistance was found in inhA gene. （4）For 137 clinical isolates tested by Spoligotyping, 104 are Mycobacterium tuberculosis with the sensitivity of 100.0%(104/104), 7 are Mycobacterium avium complex with the sensitivity of 77.8%(7/9), 15 are Mycobacterium intracellulare with the sensitivity of 93.8%(15/16), 1 are Mycobacterium fortuitum with the sensitivity of 0.0%(0/0), 10 are Mycobacterium chelonei with the sensibility of 100.0%(8/8) by DNA chip method, respectively. Kappa=0.95, U=30.6, P<0.05. Conclusion The drug-sensitive test by DNA chips method matched the absolute concentration method very well. RFP-resistance was related to the mutations at the sites of 531, 516, 526, 511 for rpoB gene, and INH-resistance was related to the mutations at the site of 315 for KatG gene. No mutation was found in inhA gene in INH-resistant isolates. DNA chip method might be a rapid and effective method for the detection of Mtb drug-resistant isolates and mycobacterium species.

Key words: Mycobacterium tuberculosis, Drug resistance, bacterial, Microbial sensitivity tests, Oligonucleotide array sequence analysis