Detection of <i>Mycobacterium tuberculosis</i> Using CRISPR-Cas12a Combined with Recombinase Polymerase Amplification

Objectives: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major infectious disease worldwide.Conventional diagnostic tests such as the tuberculin skin test and interferon-gamma release assay have limited sensitivity and reproducibility, particularly in immunocompromised patients.This study aimed to develop a rapid, sensitive, and specific molecular diagnostic platform by integrating recombinase polymerase amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) 12a detection system for MTB identification.Methods: Primers and guide RNAs (gRNAs) were designed to target the MTB-specific insertion sequence (IS) 6110.The RPA reaction amplified the target DNA at 37C-42C, and the amplified product was subsequently recognized by the Cas12a-gRNA complex.Upon target binding, Cas12a cleaved fluorophore-quencher-labeled reporter DNA, producing a detectable fluorescence signal.Reaction conditions, including enzyme and gRNA concentrations, were optimized to enhance assay performance. Results:The optimized RPA-CRISPR-Cas12a assay detected MTB genomic DNA down to 10 fg/L with no cross-reactivity against non-tuberculous bacteria.The assay generated measurable fluorescence within 40 minutes under isothermal conditions, demonstrating both high analytical sensitivity and specificity.Optimal detection was achieved at Cas12a concentrations of 80-100 nM with balanced Cas12a-gRNA ratios (1:1-1:2). Conclusion:The developed RPA-CRISPR-Cas12a platform provides a rapid, specific, and cost-effective method for detecting MTB.This assay overcomes the limitations of existing diagnostic tools and shows strong potential for point-of-care TB diagnostics in proof-of-concept.