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Journal of Clinical Microbiology, June 1999, p. 1714-1720, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Bum-Joon Kim,1 Seung-Hyun Lee,1 Mi-Ae Lyu,1 Seo-Jeong Kim,2 Gill-Han Bai,3 Sang-Jae Kim,3 Gue-Tae Chae,4 Eui-Chong Kim,5 Chang-Yong Cha,1 and Yoon-Hoh Kook1,*

Department of Microbiology and Cancer Research Center1 and Department of Clinical Pathology,5 Seoul National University College of Medicine, Seoul 110-799, Department of Pediatrics, Pundang CHA General Hospital, Pochun CHA Medical School, Kyonggi-do Sungnam 463-670,2 Korean Institute of Tuberculosis, Korean National Tuberculosis Association, Seoul 137-140,3 and Institute of Hansen's Disease, The Catholic University Medical College, Seoul 137-7014,4 Korea

Received 30 November 1998/Returned for modification 14 January 1999/Accepted 2 March 1999

For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta  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 of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. 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 amplified rpoB 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 of M. tuberculosis, rifampin resistance can be simultaneously determined.

* Corresponding author. Mailing address: Department of Microbiology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Phone: (82) 2-740-8313. Fax: (82) 2-743-0881. E-mail: yhkook{at}plaza.snu.ac.kr.

Journal of Clinical Microbiology, June 1999, p. 1714-1720, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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