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Journal of Clinical Microbiology, July 2002, p. 2509-2512, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2509-2512.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of Isoniazid-Resistant Mycobacterium tuberculosis Strains by a Multiplex Allele-Specific PCR Assay Targeting katG Codon 315 Variation

Igor Mokrousov,1* Tatiana Otten,2 Maxim Filipenko,3 Anna Vyazovaya,1 Eugeny Chrapov,3 Elena Limeschenko,1 Lidia Steklova,4 Boris Vyshnevskiy,2 and Olga Narvskaya1

Laboratory of Molecular Microbiology, St. Petersburg Pasteur Institute, 197101 St. Petersburg,1 Laboratory of Microbiology of Tuberculosis, The Research Institute of Phthisiopulmonology, 193063 St. Petersburg,2 Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, 630090 Novosibirsk,3 City Anti-Tuberculosis Dispensary, 196158 St. Petersburg, Russia4

Received 22 February 2002/ Returned for modification 13 March 2002/ Accepted 29 April 2002

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC->ACC and ACA (Ser->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inhr strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.

* Corresponding author. Mailing address: Pasteur Institute, 14 Mira St., 197101 St. Petersburg, Russia. Phone: 7 812 233 21 49. Fax: 7 812 232 92 17. E-mail: miv{at}IM4520.spb.edu.

Journal of Clinical Microbiology, July 2002, p. 2509-2512, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2509-2512.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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