Gravity-driven Microfluidic Chip Based on RPA-CRISPR/Cas12a for the Rapid Detection of Mycobacterium tuberculosis

【Objective】 To develop a rapid and efficient method for detecting Mycobacterium tuberculosis (MTB), providing a technical solution for rapid screening of tuberculosis (TB) in grassroots units and remote areas, thereby enhancing TB prevention and control efforts. 【Method】 RPA (recombinase polymerase amplification) primers and crRNA were designed, and a gravity-driven microfluidic chip was introduced to establish an RPA-CRISPR/Cas12a on-chip detection method. The sensitivity and specificity of this method were further analyzed using samples from suspected TB patients for on-chip detection and sputum culture, comparing the established method with the sputum culture method for consistency. 【Result】 The established RPA-CRISPR/Cas12a on-chip detection method achieves MTB detection within 30 min, with a limit of detection (LOD) of 1 copy/μL. Using sputum culture identification as a reference standard, the on-chip detection method demonstrated a sensitivity of 91.11%, specificity of 94.34%, positive predictive value of 93.19%, negative predictive value of 92.59%, and accuracy of 92.86%, with a Kappa value of 0.856.【Conclusion】The developed RPA-CRISPR/Cas12a on-chip detection method presents high sensitivity and specificity, characterized by rapid, simple, and convenient. © 2025 The Editorial Office of Biotechnology Bulletin. All rights reserved.