An Innovative and Efficient Fluorescent Detection Technique for in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 10-2 × 10 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.