Development of a one-step loop-mediated isothermal amplification coupled with lateral flow dipstick (LAMP-LFD) assay for rapid and sensitive tuberculosis diagnosis

Tuberculosis (TB) remains a critical global health challenge, necessitating the development of rapid, sensitive, and field-deployable diagnostic tools to replace time-consuming culture methods and complex molecular assays. This study developed and evaluated a one-step Loop-mediated Isothermal Amplification (LAMP) assay coupled with a Lateral Flow Dipstick (LFD) targeting the IS6110 insertion sequence of the Mycobacterium tuberculosis (MTB) complex. Using a dual-labeled primer strategy (FITC and biotin) for direct visual interpretation, the LAMP reaction was optimized for temperature, MgSO 4 , and enzyme concentration, and its performance was validated using 50 double-blind clinical specimens in collaboration with the National Tuberculosis Reference Laboratory. The LAMP–LFD assay demonstrated an analytical sensitivity of 10 1 CFU/mL, which was ten-fold higher than conventional PCR (10 2 CFU/mL), and showed no cross-reactivity against non-tuberculous mycobacteria, including Mycobacterium intracellulare , Mycobacterium fortuitum , and Mycobacterium avium . In clinical evaluation, the assay correctly identified all 48 MTB-positive samples and 2 negative controls, achieving full diagnostic sensitivity and specificity in this pilot cohort within approximately 65 minutes. In conclusion, the developed one-step LAMP–LFD platform provides a rapid, robust, and highly specific solution that eliminates the need for complex instrumentation and post-amplification handling, representing a practical and scalable diagnostic tool for decentralized and resource-limited settings.