Cascade CRISPR/cas Enables More Sensitive Detection of Toxoplasma gondii and Listeria monocytogenes than Single CRISPR/cas

Foodborne pathogens represent a class of pathogenic microorganisms capable of causing food poisoning or serving as foodborne vectors, constituting a major source of food safety concerns. With increasing demands for rapid diagnostics, conventional culture-based methods and PCR assays face limitations due to prolonged turnaround times and specialized facility requirements. While CRISPR-based detection has emerged as a promising rapid diagnostic platform, its inherent inability to detect low-abundance targets necessitates coupling with isothermal amplification, thereby increasing operational complexity. In this study, we preliminarily developed a novel amplification-free Cascade-CRISPR detection system utilizing a hairpin DNA amplifier. This method achieves detection sensitivity as low as 10 fM (82 parasites/μL) for DNA targets within 30 min without requiring pre-amplification, with background signal suppression achieved through optimized NaCl concentration. Validation using artificially contaminated food samples demonstrated the platform's robust performance for both <i>Toxoplasma gondii</i> (<i>T. gondii</i>) and <i>Listeria monocytogenes</i> (<i>L. monocytogenes</i>) detection, confirming broad applicability. In summary, this study preliminarily establishes an amplification-free Cascade-CRISPR detection platform that achieves high sensitivity and rapid turnaround, demonstrating strong potential for on-site screening of foodborne pathogens.