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Journal of Clinical Microbiology, November 2000, p. 4080-4085, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection and Identification of Mycobacteria by Amplification of the Internal Transcribed Spacer Regions with Genus- and Species-Specific PCR Primers

Heekyung Park,1 Hyunjung Jang,1 Cheolmin Kim,2,* Byungseon Chung,2 Chulhun L. Chang,3 Soon Kew Park,4 and Sundae Song5

Institute for Biomedical Research, SJ-Hightech Co., Ltd.,1 and Departments of Biochemistry,2 Clinical Pathology,3 and Internal Medicine,4 College of Medicine, Pusan National University, Pusan, and Institute of Clinical Research, National Masan Tuberculosis Hospital, Masan,5 Korea

Received 29 March 2000/Returned for modification 8 May 2000/Accepted 10 July 2000

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosis complex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosis complex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.

* Corresponding author. Mailing address: Department of Biochemistry, College of Medicine, Pusan National University, #10, Amidong-1-Ga, Seogu, Pusan 602-739, Korea. Phone: 82-51-240-7725. Fax: 82-51-248-1118. E-mail: kimcm{at}hyowon.cc.pusan.ac.kr.

Journal of Clinical Microbiology, November 2000, p. 4080-4085, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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