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Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products

  • M. L. FilipenkoEmail author
  • M. A. Dymova
  • A. G. Cherednichenko
  • E. A. Khrapov
  • O. V. Mishukova
  • Ya. Sh. Schwartz
Article

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.

Key Words

Mycobacterium tuberculosis drug resistance mutations pyrazinamide pncA gene 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • M. L. Filipenko
    • 1
    Email author
  • M. A. Dymova
    • 1
  • A. G. Cherednichenko
    • 2
  • E. A. Khrapov
    • 1
  • O. V. Mishukova
    • 1
  • Ya. Sh. Schwartz
    • 2
  1. 1.Institute of Chemical Biology and Fundamental MedicineSiberian Division of the Russian Academy of SciencesNovosibirskRussia
  2. 2.Novosibirsk Research Institute of TuberculosisMinistry of Health of the Russian FederationNovosibirskRussia

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