Dual-modal LAMP platform integrating biosensor and real-time fluorescence for rapid differentiation of Mycobacterium tuberculosis complex from nontuberculous mycobacteria

ABSTRACT Mycobacterium tuberculosis complex (MTBC), the primary causative agent of pulmonary tuberculosis (PTB), shares pathological features with nontuberculous mycobacteria (NTM). Some NTM species act as opportunistic pathogens that can cause NTM lung disease, often leading to diagnostic confusion with PTB. Given the persistent global burden of tuberculosis and the rising incidence of NTM infections worldwide, there is an urgent need for rapid, highly sensitive diagnostic techniques capable of distinguishing MTBC and NTM at initial clinical presentation. In this study, we developed a novel diagnostic platform that integrates loop-mediated isothermal amplification (LAMP) with nanoparticle-based lateral flow biosensors (LFB), and real-time (RT) fluorescence analysis for simultaneous detection of MTBC and NTM. Target-specific primers were designed for IS6110 (MTBC-specific) and 16S rDNA (pan-mycobacterial), and the corresponding probes were multi-labeled: FAM-biotin/digoxigenin-biotin for LFB capture, and FAM-BHQ1/Cy5-BHQ2 for real-time fluorescence monitoring. After systematic optimization, the LFB–RT–LAMP assay performed best at 67°C for 50 min. Analytical evaluation using genomic DNA from MTB reference strain H37Rv, 22 mycobacterial strains, 13 clinically relevant non-mycobacterial pathogens, and 80 respiratory specimens showed the following: (i) a limit of detection (LoD) of 576 fg/reaction for purified MTB DNA; (ii) 100% detection rate for the tested mycobacteria; (iii) 100% specificity against non-target pathogens; (iv) 100% clinical concordance with composite diagnostic PCR-based methods; and (v) a turnaround time of <60 min for both LFB readout and real-time fluorescence monitoring. Our dual-modal LFB–RT–LAMP platform provides a diagnostic workflow for simultaneous MTBC detection (via IS6110 ) and NTM screening (via 16S rDNA) in a single assay. The method achieves a turnaround time of under 60 min, a sensitivity of 576 fg/reaction, and 100% specificity against non-mycobacterial pathogens, demonstrating potential for point-of-care use in clinical and field settings. IMPORTANCE Tuberculosis (TB) and nontuberculous mycobacteria (NTM) lung disease present with similar symptoms but require completely different treatment regimens. Rapid and accurate differentiation between these pathogens at initial presentation remains a critical unmet need, as current molecular tests primarily focus on TB and are not designed for NTM screening. In this study, we developed a simple, rapid, and equipment‑free diagnostic platform that distinguishes TB from NTM within 60 min using a lateral flow biosensor, alongside a real‑time fluorescence option for laboratory use. The assay simultaneously targets IS6110 ( Mycobacterium tuberculosis complex [MTBC]‑specific) and 16S rDNA (pan‑mycobacterial), enabling species‑level identification of MTBC and genus‑level screening of NTM in a single reaction, achieving 100% accuracy on clinical samples with a limit of detection of 576 fg (~120 genome copies) per reaction. This dual‑modal approach offers practical solutions for both well‑equipped hospitals and remote clinics, addressing a critical gap in TB and NTM diagnostics.