Development of a Multiplex Real-Time PCR for Diagnosis of Tuberculosis and Multidrug Resistance

The aim of this study was to develop a multiplex qPCR method for diagnosis of tuberculosis and multidrug resistance. The method was constructed with two multiplex qPCR mixes. Mycobacterium tuberculosis and rifampicin (RIF) resistance were assessed in the first PCR mix and isoniazid (INH) resistance in the second PCR mix. Firstly, PCR amplification conditions and concentration of primers and probes were optimized. Then, analytical sensitivity/limit of detection (LOD), analytical specificity, repeatability, accuracy, and effectiveness on clinical samples were analyzed. The LOD value was determined as 124 cfu/mL. In studies conducted on 273 clinical samples, RIF and INH resistance was detected with ≥ 99% sensitivity and specificity. No false positive or negative results were obtained in the accuracy studies. In intra-experiment, inter-experiment, and inter-lot repeatability studies, the coefficient of variation was calculated as between 0.36 and 5.3%. It was also determined that the primers/probes used in this assay did not cross-react with microorganisms other than M. tuberculosis complex. In conclusion, the method optimized in the current study has high performance characteristics indicating that it can be used as an alternative for currently available molecular methods for rapid and accurate diagnosis of pulmonary tuberculosis and multidrug resistance without needing any specific equipment. Further study with different clinical samples will be useful to provide more data regarding routine utility of this method.