In House Triplex Molecular Beacon (MB) Real-time PCR Assay for Detection of Mutations Associated with Rifampicin and Isoniazid-Resistant Mycobacterium tuberculosis

Background and Objective: The emergence of Mycobacterium tuberculosis strains resistant to the two most effective first-line drugs, isoniazid and rifampicin, demands for development of a rapid and sensitive detection method for the effective treatment and to prevent dissemination of resistant strains. In the present study, using molecular beacon(MB) we developed a multiplex real-time PCR assay for simultaneous and rapid detection of M. tuberculosis strains susceptible or resistance to the two most effective first-line drugs, isoniazid and rifampicin in a single tube. Methods: Three pairs of primers and MB probes targeting katG codon 315, rpoB 81-bp codons 526 and 531 and inhA promoter region of M. tuberculosis strain H37Rv genome were used and validated by a panel of 60 clinical isolates harboring bacilli susceptible and resistant to isoniazid and/or rifampicin. For specificity testing, DNA extracted from bacterial species other than M. tuberculosis, including M. africanum, M. microti, M. canetti, M. kansassii were amplified by the same sets of primers and the probes. Results: The limit detection of the MB assay was as low as 50 DNA copies/reaction mixtures. Little or no fluorescent signals were observed when amplification was performed using DNA extracted from other Mycobacterium species. Compared to drug susceptibility test (DST) results, for rifampicin resistance, the sensitivity of the assay was 91.6% and the specificity was 100%. For isoniazid resistance, overall the sensitivity was 87.5% and the specificity was 100%. Conclusion: Overall, single-tube real-time PCR assay with molecular beacon is able to rapidly detect M. tuberculosis strains susceptible or resistant to rifampicin and isoniazid antibiotics. However, the mutants which did not have genetic alterations in the target gene sequences were not detected. Therefore, including of other genetic targets would improve the sensitivity.

کلیدواژه ها:

Isoniazid ، Molecular Beacon ، Mycobacterium tuberculosis ، Real-Time Polymerase Chain Reaction ، Rifampicin.