Amplification-Based CRISPR/Cas12a Biosensor Targeting the COX1 Gene for Specific Detection of Porcine DNA

We propose a CRISPR/Cas12a-mediated recombinase polymerase amplification (RPA) detection method that combines RPA with Cas12a cleavage for the detection of halal food adulteration, which is of global concern, particularly for Muslim consumers. We optimized the reagent concentrations for the Cas12a cleavage steps and designed and screened gRNA targeting a conserved area of the mitochondrial cytochrome C oxidase subunit I (COX1) gene. This procedure successfully detected the presence of porcine components as low as 5 pg/μL in the linear range of 5-1000 pg/μL. The assay's detection limit was 500 times lower than CRISPR-based approaches that exclude a preamplification step, allowing the detection of trace porcine DNA in food samples. The assay additionally showed no cross-reaction with nontarget species. Therefore, this detection platform shows tremendous potential as a method for the quick, sensitive, and specific detection of porcine-derived components.