分枝杆菌临床分离株菌种的分子鉴定

Molecular identification of Mycobacterium clinically isolated strains of bacteria

目的建立对临床分离分枝杆菌菌种快速、精确鉴定的方法学平台，了解分枝杆菌菌种的分布，为结核分枝杆菌（MTB）和非结核分枝杆菌（NTM）病临床精确诊断和有效治疗提供依据。方法330株分枝杆菌分离株来自于2007年5月至2008年12月在哈尔滨市胸科医院诊断为结核病的住院患者。菌株灭活后提取基因组DNA．采用PCR方法，结合PCR-限制性片段长度多态性（RFLP）和DNA序列测定方法进行菌种精确鉴定。结果330株临床分离菌株全部精确鉴定，其中328株[占99．4％（328／330）]为结核分枝杆菌复合群（MTBC），2株[占0．6％（2／330）]为NTM。328株MTBC中，MTB326株、非洲分枝杆菌（M.African）1株、田鼠分枝杆菌（舱microti）1株，分别占MTBC的99．4％（326／328）、O．3％（1／328）和0-3％（1／328）。经测序和基因组同源性分析发现，2株NTM为胞内分枝杆菌（MAC），与MAC的同源性分别为99％和93％，2株菌之间的同源性为93％。结论建立了临床分离分枝杆菌菌种快速鉴定的方法学平台，临床诊断为结核病的患者MTB比例占绝对优势。也发现有舱African、舱microti和NTM感染。

Objective To establish the methodology for identification of clinical isolates of Mycobacterium and to identify the distribution of Mycobacterium species in hospitalized patients with tuberculosis in Heilongjiang province. It would provide the basis for accurate diagnosis of infections with Mycobacterium tuberculosis （MTB） and non-tuberculous Mycobacterium （NTM） as well as for effective therapy. Methods Three hundred and thirty Mycobacterium isolates from 330 tuberculosis patients hospitalized and clinically diagnosed in Harbin Chest Hospital from May 2007 to December 2008 were collected. Genomic DNA from the isolates was extracted after inactivation of Mycobacterium. Molecular identification was carried out using PCR, PCR-restriction fragment length polymorphism （RFLP） and sequencing. Results Among 330 clinical isolates, 328 were identified as MTB complex （MTBC）, accounting for 99.4% （328/330）; 2 were NTM, accounting for 0.6% （2/330）. Among 328 MTBC isolates, 326 were MTB, one was Mycobacterium Africanum（M. African） and one was Mycobacterium microti（M, microti）, accounting for 99.4%（326/328）, 0.3%（1/328） and 0.3% （1/328）, respectively. It was found that the homology between the two NTM isolates and Mycobacterium avium intracellulare （MAC）was 99% and 93%, respectively, suggesting that the two NTM isolates were MAC. The homology between the two NTM isolates was 93%. Conclusions PCR plus PCR-RFLP and sequencing provides an ideal method for precise identification of Mycobacterium species. In the clinically diagnosed tuberculosis patients, the predominant Mycobacterium species is MTB, however M. African, M. microti as well as NTM are also found.