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Evaluation of Point Mutation Detection in Mycobacterium tuberculosis with Isoniazid Resistance Using Real-Time PCR and TaqMan Probe Assay

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Abstract

Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INH-susceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in −15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb.

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Acknowledgments

The authors are grateful to Reza Derakhshan, Rosita Vakili, and Mehdi Aganj, Medical School, MUMS, for reading the article and their valuable advice.

Author Contribution

RF performed the tests, DM was the advisor of the study and helped in designing the study, MA compiled the results and prepared the draft, SS compiled the results and prepared the draft, and RSA designed the study and proofread the manuscript.

Conflict of Interest

None declared.

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Authors and Affiliations

  1. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    F. Riahi, M. Derakhshan, A. Mosavat & S. Soleimanpour

  2. Inflammation and Inflammatory Diseases Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

    S. A. Rezaee

Authors
  1. F. Riahi
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  2. M. Derakhshan
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  3. A. Mosavat
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  4. S. Soleimanpour
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  5. S. A. Rezaee
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Corresponding author

Correspondence to S. A. Rezaee.

Additional information

A. Mosavat and S. Soleimanpour contributed equally in this study.

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Riahi, F., Derakhshan, M., Mosavat, A. et al. Evaluation of Point Mutation Detection in Mycobacterium tuberculosis with Isoniazid Resistance Using Real-Time PCR and TaqMan Probe Assay. Appl Biochem Biotechnol 175, 2447–2455 (2015). https://doi.org/10.1007/s12010-014-1442-9

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  • DOI: https://doi.org/10.1007/s12010-014-1442-9

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