Development of Mycobacterium tuberculosis Diagnosis by Multiplex Real-time PCR

Mycobacterium tuberculosis Diagnosis by Multiplex Real-time PCR

Abstract

         Tuberculosis is a major public health concern in Thailand, primarily caused by bacteria in the Mycobacterium tuberculosis complex (MTC), of which M. tuberculosis (MTB) is the most common. Non-tuberculous mycobacteria (NTM) often cause opportunistic infections, and distinguishing between these two groups of bacteria is critical for planning effective patient treatment. Real-time PCR is a diagnostic method that provides rapid, sensitive, and specific results, and can be processed for large numbers of samples. In this study, a multiplex real-time PCR method for detecting MTB/NTM, using primers and a probe specifi c to the IS6110 gene for MTC was developed, which could detect the standard MTB strain at a minimum concentration of 0.12 copies/reaction. Additionally, primers and a probe specific to the 16S rRNA gene for Mycobacterium species (PAN) were tested on standard strains of MTB and NTM, resulting in detection limits of 1.2 copies/reaction and 0.12 CFU/reaction, respectively. Evaluation of this developed method showed no cross-reactivity with other respiratory pathogens. In comparison with the gold standard culture method on 230 sputum samples, of which 90 were MTB positive and 50 were NTM-positive, the developed method detected 80 MTB-positive and 42 NTM-positive samples. Among 90 culture-negative samples, the developed method provided negative results for both MTB and NTM in 90 and 87 samples, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for MTB were 88.9%, 100.0%, 100.0%, and 93.3%, respectively, and for NTM were 84.0%, 98.3%, 93.3%, and 95.7%, respectively. This newly developed method is efficient for routine diagnostics, offering high sensitivity and specificity while reducing costs associated with importing reagents. It strengthens tuberculosis screening capabilities and supports the sustainable goal of ending tuberculosis in Thailand.