Abstract
African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe(3)O(4)@SiO(2) core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10(4) copies/μL and a limit of quantification (LOQ) of 4.2 × 10(5) copies/μL, with a dynamic range spanning 10(5) to 10(10) copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10(5) copies/μL of ASFV DNA even with high mutant concentrations (10(13) copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.