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.

FIG. 1.

Schematic view of the rpoB gene fragment targeted by the allele-specific PCR assays. Short arrows depict the primers, long double-headed arrows represent PCR fragments, either invariable (249-bp) or allele-specific (167, 181, or 214 bp); lengths not to scale. The targeted rpoB codons are in shaded boxes; the mutated bases are underlined.

FIG. 2.

Profiles generated by single-step MAS-PCR assay with purified DNA preparations from clinical M. tuberculosis strains targeting three rpoB codons: codon 531 (A), codon 526 (B), and codon 516 (C). Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC); 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).

FIG. 3.

Profiles generated by two-step nested allele-specific PCR assays with sputum slides DNA preparations. (A) First-step PCR with outer rpoB derived primers. (B to D) Analysis of three rpoB codons, codons 531, 526, and 516, respectively, by allele-specific PCR assays. Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC), 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).