Use of restriction enzyme analysis of amplified DNA coding for the hsp65 gene and polymerase chain reaction with universal primer for rapid differentiation of mycobacterium species in the clinical laboratory

Abstract

Two rapid procedures, restriction enzyme analysis of the amplified segment of the gene encoding for the 65000 mol. wt heat shock protein and a polymerase chain reaction with single universal primer (UP-PCR), were used for the identification of Mycobacterium tuberculosis complex (n = 47) and proving the species identity of non-tuberculous mycobacteria (NTM, n=36) cultured from clinical samples by comparing the resulting DNA banding pattern with patterns derived from mycobacterial type strains (n = 24). UP-PCR assay provided a rather wide limit of tolerance for variations in procedure. Although mycobacterial strains were found to generate species-specific banding patterns in both assays, M. tuberculosis and M. bovis strains and isolates produced nearly the same DNA patterns, which were very distinctive from that of all NTM tested. Investigation of the majority of M. fortuitum (n = 14) and M. kansasii (n = 7), mycobacteria most frequently causing mycobacterioses in the region, as well as other NTM isolates, showed reproducible patterns characteristic of corresponding type strains. Both methods combine the advantages of ordinary PCR and PCR 'fingerprinting', namely, the species-specific DNA pattern and primers applicable to different species. They may be applied as rapid tests for proving the identity of Mycobacterium species in a clinical laboratory.