Abstract: Objective To detect rpoB gene mutation of rifampin-resistant Mycobacterium tuberculosis insputum specimens by nested PCR-DNA sequencing,and to evaluate its value in clinical applications to develop arapid method for the detection of rifampin-resistance in Mycobacterium tuberculosis.Method One handred and twelve sputumspecimens from the patients with active pulmonary tuberculosis and 20 sputum specimens from the patients withnon-tuberculous pulmonary disease were detected by nested PCR-DNA sequencing,smear,culture,and speciesidentification.Results Thirty-one of 112 sputum specimens from the patients with active pulmonarytuberculosis had mutations of rpoB gene in M.tuberculosis by nested PCR-DNA sequencing.Of 32 sputumspecimens with rifampin-resistant strains,29 had rpoB mutations,and the mutation rate was 90.6%(29/32).2 of 39 sputum specimens with smear and culture negative had rpoB mutations.No mutation was found in 37 sputumspecimens with rifampin-sensitive strains(specificity 100%). Conclusion Nested PCR-DNA sequencing maybecome an accuracy,specific,rapid method for direct detection of rpoB gene mutation in Mycobacteriumtuberculosis from clinical specimens.

Key words: Nested PCR, DNA sequencing, Mycobacterium tuberculosis, rifampin, Drug-resistance,microorganism