Lyophilized LAMP Assay Combined with Lateral Flow Biosensor for Cold-Chain-Free Detection of Mycobacterium tuberculosis.

To decentralize tuberculosis surveillance in resource-limited and tropical regions, a rapid, robust, and cold-chain-independent near-point-of-care biosensor is urgently required to overcome the instability of conventional liquid-formulation diagnostics. Herein, we developed a ready-to-use lyophilized reaction mix Loop-Mediated Isothermal Amplification (LAMP) matrix integrated with a Lateral Flow Biosensor (LFB) for the instrument-free visual detection of Mycobacterium tuberculosis (M. tuberculosis). The freeze-dried reagents, stabilized with an optimized trehalose-BSA matrix, were hermetically packaged and systematically evaluated for long-term thermal stability across four regulated profiles (-20 °C, 4 °C, 25 °C, and 37 °C) over a 12-month period. Diagnostic viability was further validated in a double-blinded study using 191 clinical sputum specimens against gold-standard culture and line probe assays. The stabilized platform demonstrated an analytical sensitivity down to 102 CFU/mL and maintained full functional activity for up to 12 months at -20 °C and 4 weeks at 4 °C, with robust short-term resilience under tropical stress (25 °C) for up to 1 week. However, the formulation was unable to withstand prolonged exposure to harsh environmental conditions at 37 °C, resulting in a progressive decline in amplification performance following phase-transition instability. Crucially, clinical validation yielded an outstanding diagnostic sensitivity of 98.92% and a positive predictive value (PPV) of 100.00%. These findings validate the lyophilized LAMP-LFB platform as a highly reliable, sustainable, and distributable molecular diagnostic tool capable of overcoming the cold-chain bottleneck in field-based tuberculosis detection. Although the developed platform shows strong potential for near-point-of-care deployment, further optimization of the lyophilization formulation and process will be necessary to enable prolonged storage and transportation under ambient-temperature conditions.