Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products
- Authors: Filipenko M.L.1, Dymova M.A.1, Cherednichenko A.G.2, Khrapov E.A.1, Mishukova O.V.1, Schwartz Y.S.2
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Affiliations:
- Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences
- Novosibirsk Research Institute of Tuberculosis, Ministry of Health of the Russian Federation
- Issue: Vol 168, No 2 (2019)
- Pages: 264-269
- Section: Article
- URL: https://journals.rcsi.science/0007-4888/article/view/242396
- DOI: https://doi.org/10.1007/s10517-019-04688-6
- ID: 242396
Cite item
Abstract
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.
About the authors
M. L. Filipenko
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences
Author for correspondence.
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk
M. A. Dymova
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk
A. G. Cherednichenko
Novosibirsk Research Institute of Tuberculosis, Ministry of Health of the Russian Federation
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk
E. A. Khrapov
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk
O. V. Mishukova
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk
Ya. Sh. Schwartz
Novosibirsk Research Institute of Tuberculosis, Ministry of Health of the Russian Federation
Email: mlfilipenko@gmail.com
Russian Federation, Novosibirsk