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Journal of Clinical Microbiology, October 2003, p. 4630-4635, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4630-4635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of a Point Mutation Associated with High-Level Isoniazid Resistance in Mycobacterium tuberculosis by Using Real-Time PCR Technology with 3'-Minor Groove Binder-DNA Probes

H. Rogier van Doorn,1,2* Eric C. J. Claas,2 Kate E. Templeton,2 Adri G. M. van der Zanden,3 Arianne te Koppele Vije,3 Menno D. de Jong,1 Jacob Dankert,1 and Ed J. Kuijper1,2

Department of Medical Microbiology, Academic Medical Center, Amsterdam,1 Center of Infectious Diseases, Leiden University Medical Center, Leiden,2 Department of Medical Microbiology, Gelre Hospitals, Apeldoorn, The Netherlands3

Received 19 March 2003/ Returned for modification 18 May 2003/ Accepted 5 July 2003

Tuberculosis remains one of the leading infectious causes of death worldwide. The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health threat. Resistance to isoniazid (INH) is the most prevalent form of resistance in M. tuberculosis and is mainly caused by mutations in the catalase peroxidase gene (katG). Among high-level INH-resistant isolates (MIC >= 2), 89% are associated with a mutation at codon 315 of katG. There is a need to develop rapid diagnostic tests to permit appropriate antibiotic treatment and to improve clinical management. Therefore, a single-tube real-time PCR, using a novel kind of probe (3'-minor groove binder-DNA probe), was developed to detect either the wild-type or the mutant codon directly in Ziehl-Neelsen-positive sputum samples. The detection limit of the assay for purified DNA was 5 fg per well (one mycobacterial genome), and with spiked sputum samples, it was 20 copies per well, corresponding to 103 mycobacteria per ml of sputum. Sputum samples from 20 patients living in Kazakhstan or Moldova and infected with monodrug- or multidrug-resistant M. tuberculosis and 20 sputum samples from patients infected with INH-susceptible M. tuberculosis were tested. The sensitivities and specificities of the probes were 70 and 94% for the wild-type probe and 82 and 100% for the mutant probe. Binding to either probe was nonambiguous. This real-time PCR allows the rapid identification of a mutant katG allele and can easily be implemented in a clinical microbiology laboratory.

* Corresponding author. Mailing address: Department of Medical Microbiology, Academic Medical Center, P.O. Box 22700, 1100DE Amsterdam, The Netherlands. Phone: 31 20 5664860. Fax: 31 20 6979271. E-mail: h.r.vandoorn{at}amc.uva.nl.

Journal of Clinical Microbiology, October 2003, p. 4630-4635, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4630-4635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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