title: Evaluation of point mutation detection in Mycobacterium tuberculosis with isoniazid resistance using real-time PCR and TaqMan probe assay.
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.
authors.affiliation: Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: F
authors.initials: F
authors.lastname: Riahi
authors.forename: M
authors.initials: M
authors.lastname: Derakhshan
authors.forename: A
authors.initials: A
authors.lastname: Mosavat
authors.forename: S
authors.initials: S
authors.lastname: Soleimanpour
authors.forename: S A
authors.initials: SA
authors.lastname: Rezaee
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000737:chemistry; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D019914:Taq Polymerase; D007538:Isoniazid