Mycobacterium tuberculosis remains a major global public health threat, imposing significant socioeconomic burdens. Current molecular diagnostics rely heavily on nucleic acid extraction and specialized equipment, which limits their use in resource-limited settings. We developed a rapid, sensitive platform suitable for point-of-care detection by integrating FTA cards with ERA/RT-ERA amplification and CRISPR/Cas12a, enabling direct analysis of samples without conventional nucleic acid purification. Leveraging FTA cards for efficient nucleic acid release and stable preservation, we established assays targeting IS6110, esxB, gyrB, and the RNA target ASdes. Integration of FTA-based sample processing, ERA/RT-ERA amplification, and Cas12a detection enables fast and reliable identification of nucleic acid markers. The detection limits were 12.5 pM for IS6110, 6.25 pM for esxB, 25 pM for gyrB, and 12.5 pM for ASdes. Our results showed strong concordance between endpoint fluorescence and qPCR results, demonstrating robust quantitative performance for both DNA and RNA targets. In clinical serum samples, the fluorescence readouts and lateral flow strip outputs showed strong concordance with quantitative PCR results across all four targets. Collectively, the FTA-ERA-CRISPR/Cas12a platform serves as a practical and offers a reliable approach for rapid tuberculosis diagnosis at the point of need. This integrated multiple distinct targets strategy provides a versatile framework for portable molecular diagnostics and holds promise for improving TB case detection in resource-limited settings.
