Background Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, PCR-restriction fragment length polymorphism of the hsp65 gene was used to characterize the isolated mycobacteria from clinical specimens in comparison with classical biochemical method.

Methods Mycobacterial species of 4892 suspicious tuberculous patients were identified based on biochemical tests. Forty eight mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. A 439 bp PCR product of hsp65 in all selected isolates were amplified and digested with the BstEII and HaeIII restriction enzymes. The RFLP patterns were compared with GelcomparII software and revealed the species identification grouping.

Results According to the biochemical tests, a total of 229 mycobacterial isolates were identified as M. tuberculosis (183), M. bovis (14), and NTM (32). All of the 48 mycobacterial selected isolates including 16 M. tuberculosis, one M. bovis and all 32 isolates of NTM strains yielded detectable PCR product for hsp65 gene and the PCR-RFLP analysis, revealed 10 different species among NTM isolates.

Conclusion In order to identify more rapidly a wide variety of mycobacteria, the PCR-restriction fragment length polymorphism analysis of hsp65 procedure was applied. The PRA test among NTM isolates indicated that the most frequent mycobacterial strains were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. Our results showed that this method in comparison with classical methods is rapid and accurate enough for the identification of mycobacterial species from LJ culture isolates.