title: Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.
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.
authors.affiliation: Laboratory of Molecular Microbiology, St Petersburg Pasteur Institute, 197101 St Petersburg, Russia. miv@IM4520.spb.edu
authors.forename: Igor
authors.initials: I
authors.lastname: Mokrousov
authors.forename: Tatiana
authors.initials: T
authors.lastname: Otten
authors.forename: Boris
authors.initials: B
authors.lastname: Vyshnevskiy
authors.forename: Olga
authors.initials: O
authors.lastname: Narvskaya
mesh_terms: D000483:Alleles; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000904:Antibiotics, Antitubercular; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin