One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella

Salmonella is one of the most prevalent and highly transmissible food-borne pathogens, making rapid and accurate screening essential for safeguarding human health and ensuring food safety. This study introduces a one-tube nested PCR mediated CRISPR-Cas12a for ultrasensitive visual screening of Salmonella spp. using fluorescent lateral flow strip. By leveraging the simultaneous dual-segment amplification capability of the designed one-tube nested PCR and the collateral activated trans -cleavage activity of CRISPR-Cas12a, the method achieves a detection limit of ‌10 ¹ CFU/mL‌, with no cross-reactivity against other common food-borne pathogens. This approach employs the fluorophore-labeled DNA reporters that are cleaved by activated Cas12a, allowing for rapid and on-site visualization of detection results. Validation in different food matrices yields satisfactory results, demonstrating robustness against matrix interference. Comparative analysis revealed a ‌10-fold sensitivity improvement‌ over traditional single-primer PCR protocols, attributed to the dual amplification efficiency of designed one-tube nested PCR and the collateral activated cleavage specificity of CRISPR-Cas12a. The portability, rapid visual readout, and ultrasensitive performance of the method enable real-time, on-site screening of Salmonella in diverse food supply chains, even in resource-limited settings. Its high specificity, robustness against matrix effects, and minimal equipment requirements make it a transformative, user-friendly tool for enhancing global food safety surveillance and preventing outbreaks.