Development of a molecular beacon-based real-time polymerase chain reaction kit for the diagnosis of tuberculosis and drug resistance

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the fatal epidemic diseases that has threatened global public health since ancient times. Treatment of TB has become even more difficult with the emergence of Multidrug Resistant (MDR) and Extensively Drug Resistant (XDR) strains of M. tuberculosis. In order to control the disease, a rapid diagnosis and drug resistance must be determined. The aim of this thesis is to develop a multiplex qPCR diagnostic kit that can diagnose MDR-TB strains by detecting the presence of M. tuberculosis in clinical samples as well as mutations that cause RIF, INH and linezolid resistance. The kit was designed to detect M. tuberculosis and RIF resistance in the first PCR mix, and to detect INH and linezolid resistance in the second PCR mix. Following the optimization studies, kit's Analytical Sensitivity/Limit of Detection (LOD), Analytical Specificity, Reproducibility, Robustness and Effectiveness on clinical samples were analyzed. The LOD value of the kit in the diagnosis of TB was determined as 124 cfu/mL. In studies conducted with clinical samples, RIF, INH and linezolid resistance was detected with ≥ 99% sensitivity and specificity. No false positive or negative results were obtained in the kit's robustness studies. In intra-experiment, inter-experiment and inter-lot repeatability studies, the coefficient of variation of the kit was calculated as below 1%. It was also determined that the primers/probes used in the kit did not cross-react with microorganisms other than M. tuberculosis complex. As a result, the developed kit will make a significant contribution to the treatment and control of TB by detecting anti-TB drug resistance in less than 2 hours, as well as the early diagnosis of tuberculosis.