Volume 95, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant . Two multiplex real-time polymerase chain reaction (qPCR) assays were developed to detect the most common resistance-associated mutations to isoniazid (S315T, -15C → T), and rifampicin (H526Y and S531L). To assess the species specificity of the qPCR, we selected 31 nontuberculous mycobacteria (NTM) reference strains belonging to 17 species from the public collection of mycobacterial cultures (BCCM/ITM). Additionally, we tested 17 isoniazid and/or rifampicin-resistant strains with other mutations in the target genes to assess mutation specificity. The limit of detection for all the targeted mutations was 20 bacilli/reaction. Multiplex 1 showed 90%, 95%, and 100% efficiency for wild type (WT), Mut 315T, and Mut 531L, respectively; whereas Multiplex 2 showed 97%, 94%, and 90% efficiency for WT, Mut -15, and Mut 526Y, respectively. Three of 17 strains that presented other mutations in the target genes were identified as rifampicin resistant and only 3/31 NTM showed a similar melting temperature to L531 and/or T315 mutants. Thus, our proposed cascade of specific tuberculosis detection followed by drug resistance testing showed sensitivities for 315T, 531L, 526Y, and -15 detection of 100%, 100%, 100%, and 96%, respectively; and specificities of 98%, 95%, 100%, and 100, respectively.


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  • Received : 16 Feb 2016
  • Accepted : 25 Aug 2016
  • Published online : 07 Dec 2016

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