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Tuberculosis imposes a major burden of death and disease on the human population. Each year, tens of millions of people become infected with Mycobacterium tuberculosis , several millions develop clinical disease, and more than 2 million die of tuberculosis (1)(2). The successful treatment of tuberculosis depends heavily on timely diagnostics and selection of an adequate treatment strategy. Use of genotyping tools based on molecular techniques decreases the time necessary for the detection of drug resistance in M. tuberculosis from several weeks to a few days or even less, and a patient’s treatment regimen can be adjusted more rapidly to account for any detected drug resistance. Several mutations conferring resistance to the first-line drugs used for antituberculosis treatment have been described [for a review, see Ref (3)], making detection of these mutations by molecular biology techniques feasible. Rifampin and isoniazid belong to the first-line drugs used for treatment of tuberculosis (4). Resistance to rifampin, one of the most efficient antitubercular drugs, is conferred mainly by mutations in the rpoB gene. Ninety-six percent of rifampin-resistant cases are attributable to point mutations, insertions, or deletions located in an 81-nucleotide segment of the rpoB gene (5). Although resistance to isoniazid can be conferred by mutations in several different genes, mutations in katG represent the most frequent cause of the resistance (3)(6). Here we describe the allele-specific depletory PCR (ASD-PCR) assay, which was developed for the detection of mutations associated with M. tuberculosis resistance to first-line drugs, specifically isoniazid and rifampin.The accuracy and reproducibility of classic allele-specific PCR assays depend greatly on the careful selection of amplification conditions, primer design, and template concentration. Elongation through the mismatched duplex leads to accumulation of mutant template fully complementary to the initially mismatched primer, yielding the false-positive amplicon. Depending on the nature of …

New PCR-Based Assay for Detection of Common Mutations Associated with Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis