CRISPR/Cas12a-mediated Enzymatic recombinase amplification for rapid visual quantitative authentication of halal food

Economically motivated adulteration (EMA) has become a concern in food safety. We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) that integrates Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to detect halal food adulteration. We designed and screened crRNA targeting CLEC, a porcine-specific nuclear single-copy gene, and optimized the reagent concentrations and incubation times for the ERA and Cas12a cleavage steps. CAMERA was highly specific for pork ingredients detection. The DNA concentration and fluorescence signal intensity relationship was linear at DNA concentrations of 20-0.032 ng/mu L. CAMERA detected as few as two CLEC copies and quantified samples with porcine DNA content as low as 5% within 25 min. The system could be operated in a miniaturized working mode that requires no technical expertise or professional equipment, making CAMERA a valuable tool in resource-limited areas for the qualitative and quantitative detection of pork ingredients in halal food.

Automated summary

We propose a CRISPR/Cas12a Mediated Enzymatic Recombinase Amplification detection system (CAMERA) for the detection of adulteration in pork ingredients in halal food. CAMERA combines Enzymatic Recombinase Amplification (ERA) and Cas12a cleavage to achieve high specificity and sensitivity. It can detect samples with as few as two copies of porcine-specific gene and quantify samples with as low as 5% porcine DNA content within 25 minutes. The system does not require technical expertise or professional equipment, making it a valuable tool for qualitative and quantitative detection in resource-limited areas.