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Journal of Clinical Microbiology, July 2001, p. 2610-2617, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2610-2617.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputa by Nested PCR-Linked Single-Strand Conformation Polymorphism and DNA Sequencing

Bum-Joon Kim,1 Keun-Hwa Lee,2 Bo-Na Park,2 Seo-Jeong Kim,3 Eun-Mi Park,4 Young-Gil Park,4 Gil-Han Bai,4 Sang-Jae Kim,4 and Yoon-Hoh Kook2,*

Department of Microbiology, Cheju National University College of Medicine, Cheju 690-756,1 Department of Microbiology and Institute of Endemic Diseases, Medical Research Center, Seoul National University College of Medicine, and Clinical Research Institute, Seoul National University Hospital, Seoul 110-799,2 Department of Pediatrics, Pundang CHA General Hospital, Pochun CHA Medical School, Kyonggi Sungnam 463-670,3 and The Korean Institute of Tuberculosis, the Korean National Tuberculosis Association, Seoul 137-140,4 Korea

Received 6 November 2000/Returned for modification 4 February 2001/Accepted 26 April 2001

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

* 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.

Journal of Clinical Microbiology, July 2001, p. 2610-2617, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2610-2617.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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