Format

Send to

Choose Destination
See comment in PubMed Commons below
Antimicrob Agents Chemother. 2003 Jul;47(7):2231-5.

Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

Author information

1
Laboratory of Molecular Microbiology, St Petersburg Pasteur Institute, 197101 St Petersburg, Russia. miv@IM4520.spb.edu

Abstract

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.

PMID:
12821473
PMCID:
PMC161874
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Support Center