Toehold-Containing Three-Way Junction-Initiated Multiple Exponential Amplification and CRISPR/Cas14a Assistant Magnetic Separation Enhanced Visual Detection of Mycobacterium Tuberculosis

Rapid and simple nucleic acid detection is significant for disease diagnosis and pathogen screening, especially under specific conditions. However, achieving highly sensitive and specific nucleic acid detection to meet the time and equipment demand remains technologically challenging. In this study, we proposed a magnetic separation enhanced colorimetry biosensor based on a toehold-containing three-way junction (TWJ) induced multiple isothermal exponential amplification and the CRISPR/Cas14a (C-TEC) biosensor. The TWJ template was designed as a Y-X-Y structure. In the presence of the target, the formation of toehold-containing TWJ complex induced primer extension, leading to the generation of amplified single-stranded DNA; this amplified DNA could then bind to either the free TWJ template for EXPAR reaction or the toehold of the TWJ complex for toehold-mediated strand displacement, thereby enabling the recycling of the target. The amplification products could trigger CRISPR/Cas14a for efficient trans-cleavage and release the magnetically bound gold nanoparticle probes for colorimetry detection. Using Mycobacterium tuberculosis 16S rDNA as the target, the proposed C-TEC could detect 16S rDNA down to 50 fM by the naked eye and 20.71 fM by UV-vis detector at 520 nm within 90 min under optimal conditions. We successfully applied this biosensor to clinical isolates of Mycobacterium tuberculosis. In addition, the C-TEC biosensor also showed feasibility for the detection of RNA viruses. In conclusion, the proposed C-TEC is a convenient, fast, and versatile platform for visual detection of pathogen DNA/RNA and has potential clinical applications.