Molecular identification of Mycobacterium clinically isolated strains of bacteria

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,326were 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. Key words: Mycobacterium; Species identification; Polymerase chain reaction; Sequence alignment