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 Toxoplasma gondii (T. gondii) and Listeria monocytogenes (L. monocytogenes) 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.