Plasma-based ultrasensitive detection of Mycobacterium tuberculosis ESAT6/CFP10 fusion antigen using a CRISPR-driven aptamer fluorescence testing (CRAFT)

Tuberculosis (TB) screening in clinical diagnosis is challenging due to issues such as sputum dependence, time-consuming procedures, and high costs. In this study, we introduce a CRAFT (CRISPR-Driven Aptamer Fluorescence Testing), an aptamer-based CRISPR/Cas12a assay designed for the rapid and sensitive detection of Mycobacterium tuberculosis (Mtb) antigens from peripheral blood. Aptamer 3 (Ap3) and the aptamer-mediated probe (Aptamer-blocker 3–7) were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Ap3 demonstrated a dissociation constant (Kd) of 8.3E-7 M with the ESAT6/CFP10 fusion proteins (EC proteins), which are produced during the replicative phase of Mtb. Upon labeling the EC proteins with Aptamer-blocker 3–7 (Ap-blocker 3–7) probe, single-stranded DNA (ssDNA) blocker 3–7 was released, thereby completing the process for RPA-based CRISPR/Cas12a fluorescence detection. After optimizing multiple parameters, CRAFT achieved a detection limit of 0.1 ag/mL EC proteins (equivalent to 3 protein particles per mL) within 120 min from plasma sample to result. The method was validated with 86 clinical plasma samples confirmed the method's high diagnostic accuracy for Mtb infection (sensitivity: 97.1 %, 95 % confidence interval (CI) [0.849–0.998]); specificity: 98.0 %, 95 % CI [0.897–0.999]), supporting its utility in early therapeutic evaluation of tuberculosis management.