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

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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 referencestrains of mycobacteria and clinical isolates (107 strains) byPCR. The nucleotide sequences were directly determined (306 bp)and aligned by using the multiple alignment algorithm in the MegAlignpackage (DNASTAR) and the MEGA program. A phylogenetic tree wasconstructed by the neighbor-joining method. Comparative sequenceanalysis of rpoB DNAs provided the basis for species differentiationwithin the genus Mycobacterium. Slowly and rapidly growing groupsof mycobacteria were clearly separated, and each mycobacterialspecies was differentiated as a distinct entity in the phylogenetictree. Pathogenic Mycobacterium kansasii was easily differentiatedfrom nonpathogenic M. gastri; this differentiation cannot be achievedby using 16S rRNA gene (rDNA) sequences. By being grouped intospecies-specific clusters with low-level sequence divergence amongstrains of the same species, all of the clinical isolates couldbe easily identified. These results suggest that comparative sequenceanalysis of amplified rpoB DNAs can be used efficiently to identifyclinical isolates of mycobacteria in parallel with traditionalculture methods and as a supplement to 16S rDNA gene analysis.Furthermore, in the case of M. tuberculosis, rifampin resistancecan be simultaneouslydetermined.

^* 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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