طراحی روش Multiplex Allele Specific PCR برای تعیین سویه های مقاوم به افلوکساسین مایکوباکتریوم توبرکلوزیس و مقایسه آن با سایر روشهای مولکولی

Title

Multiplex Allele Specific-PCR Methods for Fast Detection of GyrA Gene Mutaions in Clinical Isolates of Mycobacterium Tuberculosis

English Abstract

Background & Objectives: Two molecular methods were designed and compared for rapid detection of resistance to ofloxacin in clinical isolates of Mycobacterium tuberculosis. Methods: From 136 M. tuberculosis clinical isolates, 41 strains were used for comparison of detection of mutations associated with resistance in gyrA gene by Allele Specific-PCR (AS PCR), Multiplex Allele Specific-PCR (MAS PCR) and Semi-nested Allele Specific-PCR (SnAS PCR). Specific internal primers were designed for detection any changes in 90, 91 and 94 codons. Sequencing methods were accomplished for evaluation of the results as gold standard. Results: AS-PCR and SnAS-PCR methods could detect mutations by formation or not formation of internal bounds. MAS-PCR by like mechanism has good performance. Totally, from 37 strains phenotypically resistant to ofloxacine 32 strains were mutant and 5 strains were non mutant that have sensitivity and specificity, 86/11% and 100% , respectively. Sequence results were concordant by results molecular methods. Conclusion: Results of the study showed that MAS-PCR methods could be use as a routine test for fast detection of resistance to fluroquinolones in M. tuberculosis

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