Integrated detection of Mycobacterium tuberculosis at point-of-care using a streamlined digital CRISPR-based platform

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major global health threat, with millions of new cases and deaths annually. Current diagnostic methods, such as sputum smear microscopy and molecular assays, suffers from limitations in sensitivity, cost, and accessibility, particularly in resource-limited settings. To address the urgent need for a sensitive, rapid, and accessible diagnostic solution, we developed dAFCas-TB, a fully integrated, digital, amplification-free CRISPR-based platform for MTB detection. This system integrates a complete workflow that includes rapid nucleic acid extraction, droplet-based digital CRISPR detection, and smartphone-assisted imaging, aiming to support point-of-care testing (POCT) in underserved regions. We evaluated three rapid nucleic acid extraction methods specifically designed to address the challenge posed by the thick, lipid-rich cell wall of MTB. Among these methods, heat-based lysis demonstrated the highest compatibility with the CRISPR assay and achieved superior performance in both extraction efficiency and detection signal compared to commercial kits. Clinical validation using 58 MTB samples demonstrated 97.7 % sensitivity and 93.8 % specificity, with a total detection time of approximately 1.5 h. Compared to the WHO-recommended GeneXpert system, dAFCas-TB offers a cost-effective alternative, making it highly suitable for TB screening in low-resource areas. This study highlights the potential of dAFCas-TB as a rapid, sensitive, and accessible diagnostic tool for early MTB detection and widespread screening. • dAFCas-TB integrates nucleic acid extraction with amplification-free digital CRISPR detection. • dAFCas-TB utilizes heat lysis to break MTB cell wall, enhancing DNA release and detection. • dAFCas-TB detects TB within 1.5 h under $1 per test, with high sensitivity and specificity.