Diffcrential diagnosis of tuberculosis and nontuberculous infectious diseases by PCR-RFLP and sequence-specific oligonucleotide probes

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Identification of tuberculous and non-tuberculous mycobacteria by biochemical methods is a long-term process which takes up to 8 weeks for completion and requires expertise for interpretation of the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers which amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ gene from 19 mycobacterial species (M.tuberculosis, M.bovis BCG,M.africanum, M.microti, M.kansasii, M.marinum, M.GASTRI, M.simiae, M.scrofulaceum, M.szulgai, M.gordonae, M.avium, M.intracellulare, M.xenopi, M.fortuitum, M.chelonei, M.hemophilum and M.paratuberculosis).On the basis of the dnaJ gene sequences, we identified the species-specific restriction sites, which allows us to differentiate most of the mycobacterial DNA by a combination of the PCR with the restriction fragment length polymorphism analysis (PCR-RFLP). The PCR assay for the dnaJ gene was sensitive and specific enough to detect the mycobacterial DNA directly in clinical materials, such as sputa, bronchoalveolar lavages or cerebrospinal fluids. Furthermore, we developed dot blot hybridization analysis using species-specific oligonucleotide probes for the M.tuberculosis complex, M.a vium, M.intracellulare and M.kansasii, allowing a rapid identification of these species following PCR for the dnaJ gene. We conclude that PCR with the genus-specific primer which amplifies the dnaJ gene and subsequent both RFLP analysis with restriction enzymes and dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and non-tuberculous mycobacterial infections.