Detection and identification of mycobacterial DNA with sputa and AIDS samples by nested-polymerase chain reaction (nested-PCR)

Abstract

We have demonstrated the nested-polymerase chain reaction (nested-PCR) assay detected from 14 mycobacterial species. We have further demonstrated the species specific restriction sites within amplified dnaJ gene, which allowed us to differentiate the mycobacterial DNA by combination of the PCR with the restriction fragment length polymorphism (PCR-RFLP). Nested-PCR-RFLP was used to detect and identified mycobacterial DNA in the sputa samples and HIV related samples. The target DNA was a 196-base pair segment of dnaJ gene. Of 68 sputum samples tested, 7 were smear positive for acid-fast bacilli and culture. The 7 samples were also identified with Mycobacterium tuberculosis by PCR-RFLP. We tested 69 HIV related samples (19 frozen samples, 22 paraffin embedded samples and 28 lymphocyte from HIV infected people). Of 38 samples were positive for nested-PCR, 8 M. tuberculosis complex, 7 M. avium, 8 M. intracellulare, and 16 others were detected by PCR-RFLP. The PCR method is useful for the rapid diagnosis of mycobacterial infection.