تشخیص سریع موتاسیون در ژن عامل مقاومت به ریفابوتین در سویه های کلینیکی مایکوباکتریوم توبرکلوزیس به روش Allele Specific PCR

Background and purpose: Molecular detection of antibiotic resistance in clinical strains of M.tuberculosis is of great importance. In this study, we developed a method for rapid detection of mutations resistant to the rifabutin antibiotic resistant gene. Material and methods: In this study 40 clinical isolates of M.tuberculosis including 12 resistant and 28 susceptible isolates to rifabutin were used. The isolates were obtained from Tuberculosis and Pediatric Infectious Research Center affiliated to Arak University of Medical Sciences. Specific primers were designed and corrected by BLAST-IDT and MEGA software. The primers for an Allele Specific PCR was able to detect the desired hot point in the gene from 3´end in 516-526-531 codons and could also reconnoiter mutant state Therefore, lack of formation of band in PCR product indicates the resistance of strain to rifabutin. Some selected samples were sequenced and compared with results derived from ASP. Results: 12 M.tuberculosis isolates resistant to rifabutin, mutations in one of the three main codons were detected in 10 strains using Allele Specific PCR method. Susceptible strains did not show any mutations in these codons. The sensitivity of the method was 80% (95% CI: 0.55-0.95) and the specificity was 100% (95% CI:0.87-1). Results of sequencing were concordant with results of ASP method. Conclusion: The results showed that Allele Specific-PCR was a rapid and simple method for fast detection of rifabutin resistance in M.tuberculosis isolates and is recommended for routine works.