Differentiation of Mycobacterial Species by PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA Polymerase Gene (rpoB)

doi:10.1128/JCM.39.6.2102-2109.2001

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Journal of Clinical Microbiology, June 2001, p. 2102-2109, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2102-2109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Differentiation of Mycobacterial Species by PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA Polymerase Gene (rpoB)

Bum-Joon Kim,¹ Keun-Hwa Lee,²^,³^,⁴ Bo-Na Park,²^,³^,⁴ Seo-Jeong Kim,⁵ Gill-Han Bai,⁶ Sang-Jae Kim,⁶ and Yoon-Hoh Kook²^,³^,⁴^,^*

Department of Microbiology, Cheju National University College of Medicine, Cheju 690-7561,¹ Department of Microbiology, Institute of Endemic Diseases, SNUMRC,² Cancer Research Center, Seoul National University College of Medicine,³ and Clinical Research Institute, Seoul National University Hospital,⁴ Seoul 110-799, Department of Pediatrics, Pundang CHA General Hospital, Pochun CHA Medical School, Kyonggi-do Sungnam 463-670,⁵ and The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140,⁶ Korea

Received 16 October 2000/Returned for modification 2 January 2001/Accepted 8 April 2001

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif^r region, was used for the differential identification of 49 mycobacteria.The DNA had been used previously for the identification of mycobacterialspecies by comparative sequence analysis (B. J. Kim et al., J.Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restrictionenzymes (HaeIII, HindII, MvaI, and AccII), which were selectedon the basis of rpoB DNA sequences, generated distinctive PRApatterns that allowed not only the reference strains but alsothe clinical isolates of mycobacteria to be distinguished. Bothrapidly and slowly growing mycobacteria were distinctly differentiatedby HaeIII digestion of the amplified rpoB DNA. By HindII digestionthe Mycobacterium tuberculosis complex was distinguished fromthe other mycobacteria. Furthermore, six subspecies of Mycobacteriumkansasii (subspecies I to VI) as well as the closely related Mycobacteriumgastri, and other closely related species, were distinguishedby simultaneous digestion of MvaI and AccII. According to therpoB PRA scheme, 240 strains of clinical isolates could be identified.It was also possible to detect and identify M. tuberculosis directlyfrom sputa and bronchoalveolar lavage specimens. These resultssuggest that PRA of rpoB DNA is a simple and feasible method notonly for the differentiation of culture isolates but also forthe rapid detection and identification of pathogenic mycobacteriain primary clinicalspecimens.

^* Corresponding author. Mailing address: Department of Microbiology, Seoul National University College of Medicine, 28 Yongon-dong,Chongno-gu, Seoul 110-799, Korea. Phone: (82) 2-740-8306. Fax:(82) 2-743-0881. E-mail: yhkook{at}plaza.snu.ac.kr.

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