Background: The emergence of drug-resistant strain of M.tuberculosis is one of the most critical issues facing TB researchers and clinicians. Rapid diagnosis of drug-resistant tuberculosis is essential for the prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. The aim of this study is to develop a Real-time PCR assay for the detection of mutations in RRDR (rifampcin resistance determinant region) of rpoB which conferring rifampicin resistance in Mycobacterium tuberculosis.

 Materials and Methods: In this experimental study, the primer and probe set were designed for a RRDR region of rpoB gene using a specialized software. Clinical specimens that had previously been evaluated resistant or sensitive by using convential method, were used for assessing the clinical sensitivity and specificity of the assay.

Results: The clinical sensitivity of the assay was determined 100%. The primers and the probes were rpoB specific and no cross-reaction was observed with other microorganisms and human genome bioinformatically. The clinical specificity of developed Real-time PCR assay was examined experimentally using 25 negative samples and determined to be 100%.

Conclusion: The developed real-time PCR assay can be used as an appropriate and efficient tool for the rapid detection of rifampicin-resistant Mycobacterium tuberculosis.