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Antimicrobial Agents and Chemotherapy, July 2003, p. 2231-2235, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2231-2235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears

Igor Mokrousov,1* Tatiana Otten,2 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, Russia2

Received 11 December 2002/ Returned for modification 10 February 2003/ Accepted 9 April 2003

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.

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

Antimicrobial Agents and Chemotherapy, July 2003, p. 2231-2235, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2231-2235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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