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We developed a protocol for detection of mutations in the pncA gene associated with M. tuberculosis resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZAR DNA isolates carrying mutations in the pncA gene, 3 PZAR isolates without mutations in the pncA gene, and 20 PZAS isolates without mutations in the pncA gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the pncA gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy — 96.55% vs. 93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software. [ABSTRACT FROM AUTHOR]

Genotyping and analysis the drug resistance of 59 isolates of M. tuberculosis obtained from patients living in Altai Territory were performed using a BACTEC MGIT 960 fluorometric system by means of VNTR typing (variable number tandem repeat), PCR-RFLP analysis, and sequence analysis. The occurrence frequency was highest for isolates of the Beijing family ( n=30, 50.8%). Analysis of mutation spectrum in the rpoB gene associated with rifampicin resistance revealed the major mutation (codon 531 of the rpoB gene) in 93% samples, which allows us to use rapid test systems. [ABSTRACT FROM AUTHOR]