Identification of the mutations related to resistance of mycobacterium tuberculosis to isoniazid by use of PCR-RFLP in TB patients

Isoniazid is one of the essential first line drugs in TB treatment. Resistance rate to this drug is increasing in many parts of the world. Mutations in KatG and inhA genes are frequently the cause of resistance of mycobacterium TB to INH. The aim of this study was to find a precise method for early detection of mutations associated with resistance of mycobacterium TB to INH. After performing sensitivity tests, presence of mutations in special loci of katG and inhA genes in 90 specimens obtained from positive cultures of TB patients was investigated. PCR-RLFP method was used to detect mutations in katG 315 codon. The PCR product resulting from the reproduction of this genetic segment (620 bp) was digested by restriction enzyme MspI. To identify mutations in inhA gene, MAS-PCR technique was used. 34.5% of the INH resistant strains had Thr315 phenotype and 65.5% had Ser 315 phenotype. Thr 315 is 100% specific for determination of INH resistant isolates. Among 52 resistant isolates, 34.6% had Arg and 65.4% had Leu in codon 463. The frequencies of mutations in the (-15C → T) locus of inhA gene were 20% and 16.7% in MDR and Non MDR isolates respectively. By using PCR-RFLP with restrictive enzyme and MAS PCR mutations in codon 315 of KatG gene and promoter location of inhA gene can be identified. These methods are simpler and cheaper than other methods and provide early accurate and reliable results.