CRISPR/Cas12-Driven Exponential Amplification Combined with a Lateral Flow Biosensor Enabling Rapid and Highly Sensitive DNA Detection

Rapid and precise detection of specific DNA is valuable for biological research and clinical disease diagnosis. Clustered regularly interspaced short palindromic repeat (CRISPR) technology can enhance existing DNA testing, making DNA detection faster, more portable, and more accurate. This study presents a new Cas12-driven exponential amplification-based lateral flow biosensor (CADEX-LF) for rapid and highly sensitive DNA detection. CADEX-LF takes full advantage of the highly specific target-loading-dependent trans-cleavage activity of Cas12 and the extremely high efficiency of nicking endonuclease-mediated exponential amplification. The adoption of lateral flow readout enables CADEX-LF for point-of-care (POC) use without requiring complicated supplementary equipment. CADEX-LF was shown to achieve a detection sensitivity of 2 × 10^–15 M within 45 min of measurement time and displayed outstanding specificity with double-base resolution. Furthermore, CADEX-LF could identify herpes simplex virus 1 (HSV-1) DNA in tears of rabbits and clinical patients with HSV-1 keratitis, exhibiting its practical application potential in clinical diagnosis. The proposed CADEX-LF biosensor may have great promise for point-of-care disease diagnosis in resource-limited environments.