Integrating Paper‐Based Microfluidics and Lateral Flow Strip into Nucleic Acid Amplification Device toward Rapid, Low‐Cost, and Visual Diagnosis of Multiple Mycobacteria

Abstract Efficient diagnosis of mycobacterial infections can effectively manage and prevent the transmission of infectious diseases. Unfortunately, existing diagnostic strategies are challenged by long assay times, high costs, and highly specialized expertise to distinguish between pulmonary tuberculosis (PTB) and nontuberculous mycobacterial pulmonary diseases (NTM‐PDs). Herein, in this study, an optimized 3D paper‐based analytical device (µPAD) is incorporated with a closed lateral flow (LF) strip into a loop‐mediated isothermal amplification (LAMP) device (3D‐µPAD‐LF‐LAMP) for rapid, low‐cost, and visual detection of pathogenic mycobacteria. The platform's microfluidic feature enhanced the nucleic acid amplification, thereby reducing the costs and time as compared to boiling, easyMAG, and QIAGEN techniques. Moreover, the LF unit is specifically designed to minimize aerosol contamination for a user‐friendly and visual readout. 3D‐µPAD‐LF‐LAMP is optimized and assessed using standard strains, demonstrating a limit of detection (LOD) down to 10 fg reaction −1 . In a cohort of 815 patients, 3D‐µPAD‐LF‐LAMP displays significantly better sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic accuracy than conventional bacterial culture and Xpert techniques. Collectively, 3D‐µPAD‐LF‐LAMP demonstrates enhanced accessibility, efficiency, and practicality for the diagnosis of multiple pathogenic mycobacteria, which can be applied across diverse clinical settings, thereby ultimately improving public health outcomes.