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For the differentiation and identification of mycobacterial species, therpoB gene, encoding the β subunit of RNA polymerase, was investigated.rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis ofrpoB DNAs provided the basis for species differentiation within the genusMycobacterium . Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. PathogenicMycobacterium kansasii was easily differentiated from nonpathogenicM. gastri ; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplifiedrpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case ofM. tuberculosis , rifampin resistance can be simultaneously determined.

Identification of Mycobacterial Species by Comparative Sequence Analysis of the RNA Polymerase Gene ( rpoB )