Analytical performance of a multi-target open real-time PCR assay for simultaneous detection of tube

Abstract Background Timely diagnosis of tuberculosis and drug resistance remains a cornerstone of effective disease control. Multiplex open molecular platforms capable of simultaneously detecting Mycobacterium tuberculosis complex (MTBc), non-tuberculous mycobacteria (NTM), and resistance to first-line anti-tuberculosis drugs could streamline diagnostic pathways. Methods We conducted a laboratory-based evaluation of two multiplex real-time PCR assays (MTBc/NTM R-Gene® and MTB-RIF/INH R-Gene®) using 300 well-characterized samples, including 150 MTBc-positive culture isolates (including rifampic

in-resistant, isoniazid-resistant, and drug-susceptible strains) and 150 MTBc-negative samples (50 NTM isolates and 100 mycobacteria-negative specimens). Composite reference standards included culture, MPT64 antigen testing, and line probe assay corroborated by phenotypic drug susceptibility testing for resistance profiling, with NTM speciation performed using a dedicated line probe assay. DNA extraction was performed using the QIAamp DNA Mini Kit (QIAGEN, Germany), followed by amplification on a real-time PCR platform according to manufacturer instructions. The diagnostic performance was asse

ssed against composite reference standards. Results The analytical performance for detecting MTBc demonstrated 100% sensitivity and specificity (150/150). NTM detection showed 70·0% sensitivity (35/50) and a specificity of 100%, highlighting limitations in coverage of NTM species. Rifampicin resistance was detected with a sensitivity of 96·0% (48/50) and specificity of 100%, whereas isoniazid resistance detection was 100% sensitive and specific (50/50). Agreement with established reference standards was high (κ=0·76–1·00) within this analytical context. Interpretation This analytical validatio