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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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          Author and article information

          Journal
          Appl Biochem Biotechnol
          Applied biochemistry and biotechnology
          Springer Science and Business Media LLC
          1559-0291
          0273-2289
          Mar 2015
          : 175
          : 5
          Affiliations
          [1 ] Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
          Article
          10.1007/s12010-014-1442-9
          25503088
          28f81c8c-73cc-4e5f-9df1-8ef3c936323a
          History

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