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Currently, tuberculosis is still one of the most common infectious diseases, mainly caused by Mycobacterium tuberculosis bacterium infection. PCR is a molecular biology technique used to detect infectious agents, especially viruses or bacteria with slowly culture as in the case of Mycobacterium tuberculosis because of its fastness, accurateness and high sensitivity. We carried out this study in order to develop a protocol based on Realtime PCR using 16S rRNA gene as a target sequence which is extensively used in taxonomy, molecular evolution and medical diagnostics. We have succeeded in designing the primers/probes on the 16S rRNA gene to detect Mycobacterium tuberculosis by Realtime PCR; have identified the parameters of the specificity of the primers/probe, theoretically and experimentally; the sensitivity of the primers/probe in the Realtime PCR protocol reached to 102 copies/ml; the protocol was also tested on 30 samples given good results.