A rapid molecular assay for the detection of Mycobacterium avium using loop-mediated isothermal amplification (LAMP) combined with a lateral flow biosensor.

Mycobacterium avium is a slow-growing nontuberculous mycobacterium (NTM) capable of infecting humans and other animals. As an opportunistic pathogen, it can cause severe diseases such as pulmonary infections and lymphadenitis in immunocompromised individuals. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow biosensor (LFB) for the rapid detection of Mycobacterium avium.LAMP is a simple, rapid, and isothermal nucleic acid amplification method, and when combined with LFB, its results can be directly observed with the naked eye. We designed a set of six primers for LAMP to amplify the Mycobacterium avium-specific insertion sequence 1311 (IS1311) gene. Isothermal amplification was performed at 68°C for 40 minutes, followed by reaction termination via incubation at 85°C for 5 minutes. The reaction products were subsequently detected using LFB, enabling visual interpretation of the results.This assay was validated against 37 pathogens, including Mycobacterium avium, the Mycobacterium tuberculosis complex, other common nontuberculous mycobacteria, and common respiratory bacteria. The validation results demonstrated high sensitivity and specificity of the assay for the detection of Mycobacterium avium. The limit of detection (LOD) of this method for pure Mycobacterium avium DNA was 100 fg/μL, and its sensitivity for sputum samples was 100 colony-forming units (CFU). Therefore, this LAMP-LFB assay provides a simple, rapid, sensitive, and specific approach for the detection of Mycobacterium avium, which holds great potential for clinical application.