PCR-RFLP Based On Hsp65and RpoB For Species Identification Of Nontuberculous Mycobacteria

Mycobacterium comprises Mycobacterium tuberculosis complex (MTBC), M. leprae and nontuberculous mycobacteria (NTM). Because both NTM and MTBC can cause pulmonary diseases and damages of other parts of the body, of which the symptoms are often similar, differential diagnosis in clinical practice is difficult. So it is important to identify species of Mycobacterium for the diagnosis and treatment. The biochemical methods based on culture for the species identification are laborious and time-consuming, sometimes even providing wrong results. With the development of molecular biology, lots of rapid, specific and highly efficient methods based on molecular biotechnology for the species identification, such as PCR-SSCP, PCR-RFLP. DNA probe, DNA chip, DNA sequence analysis, etc, have been developed and used m the identification of clinical Mycobacterium isolates. In this study, the effects of PCR-RFLP based on hsp65and rpoB for NTM species identification were evaluated using clinical Aycobacterium isolates collected in China.1Evaluation of the effects of hsp65-and/poB-PCR-RFLP for the species identification of NTM clinical isolatesNTM were differentiated from clinical isolates by PNB/TCH test and multi-locus PCR and identified to the species level by hsp65-and rpoB-PCR-RFLP, respectively, using hsp65sequence analysis as the gold standard method. The results showed that, of130clinical isolates,22strains were MTB.108strains were NTM. There was no significant difference between PNB/TCH test and multi-locus PCR (P>0.05). Multi-locus PCR needs much shorter time than PNB/TCH test. A total of108NTM were identified to species, including53(49.07%) M. intracellulare,23(21.30%) M. abscessus,19(17.59%) M. avium,4(3.70%) M. massiliense,4(3.70%) M.gordonae,1(0.93%) M. seoulense,1(0.93%) M. kumamotonense,1(0.93%) M. fortuitum,1(0.93%) M. holsaticum and1(0.93%) M. monacense. respectively. There are lots of NTM species which cause chronic lung disease in Fujian Province, and M. intracellulare, M. abscessus and M. avium are the main prevalent Mycobacterium pathogenic bacteria. Compared with hsp65sequencing, the sensitivity and specificity were99.07%,100%for hsp65PCR-RFLP and98.15%.100%for rpoB PCR-RFLP, respectively. There was no significant difference between the results of hsp65PCR-RFLP and rpoB PCR-RFLP (P>0.05).2Isolation and identification of mycobacteria from animal samplesA total of110animal samples were processed by neutral centrifuge method, and then inoculated on L-J solid media and in MIGT960liquid media. A total of29acid-fast bacilli (AFB) strains were isolated and were identified by PNB/TCH test, DNA sequencing of hsp65, hsp65-and rpoB-PCR-RFLP, multi-locus PCR and Spoligotyping.19strains were NTM, including11(57.89%) M. fortuitum5(26.32%) M. septicum.1(5.26%) M. intracellulare,1(5.26%) M. abscessus and1(5.26%) M. monacense, respectively.3strains were MTB Beijing genotype, and7strains were Nocardia farcin ica.