Detection of a Point Mutation Associated with High-Level Isoniazid Resistance in Mycobacterium tuberculosis by Using Real-Time PCR Technology with 3′-Minor Groove Binder-DNA Probes

Abstract

Tuberculosis remains one of the leading infectious causes of death worldwide. The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health threat. Resistance to isoniazid (INH) is the most prevalent form of resistance in M. tuberculosis and is mainly caused by mutations in the catalase peroxidase gene ( katG ). Among high-level INH-resistant isolates (MIC ≥ 2), 89% are associated with a mutation at codon 315 of katG . There is a need to develop rapid diagnostic tests to permit appropriate antibiotic treatment and to improve clinical management. Therefore, a single-tube real-time PCR, using a novel kind of probe (3′-minor groove binder-DNA probe), was developed to detect either the wild-type or the mutant codon directly in Ziehl-Neelsen-positive sputum samples. The detection limit of the assay for purified DNA was 5 fg per well (one mycobacterial genome), and with spiked sputum samples, it was 20 copies per well, corresponding to 10 ³ mycobacteria per ml of sputum. Sputum samples from 20 patients living in Kazakhstan or Moldova and infected with monodrug- or multidrug-resistant M. tuberculosis and 20 sputum samples from patients infected with INH-susceptible M. tuberculosis were tested. The sensitivities and specificities of the probes were 70 and 94% for the wild-type probe and 82 and 100% for the mutant probe. Binding to either probe was nonambiguous. This real-time PCR allows the rapid identification of a mutant katG allele and can easily be implemented in a clinical microbiology laboratory.