A Rapid and Sensitive RPA-CRISPR Platform for Beef Authenticity andAdulteration Detection

Introduction: Beef adulteration with cheaper meats threatens consumer trust and public health, creating a need for detection methods beyond equipment-intensive conventional techniques like qPCR and ELISA. Here, a rapid, sensitive, and field-applicable method was developed based on a portable platform integrating Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a for bovine-derived DNA identification Methods: Species-specific genes were first rapidly amplified under isothermal conditions (37-42 °C, 15-30 min) using Recombinase Polymerase Amplification (RPA). Subsequently, CRISPR/Cas12a-mediated detection was performed in a closed-tube format: target DNA activates Cas12a’s trans-cleavage activity, leading to the non-specific cleavage of single-stranded DNA (ssDNA) reporters and the production of a fluorescent signal. Results: Under the optimal conditions (reaction temperature of 37°C, incubation time of 20 min, crRNA:Cas12a ratio of 1:1, and fluorescent probe concentration of 200 nM), the assay achieves visual, equipment-free results and demonstrates high specificity for beef against pork/lamb and sensitivity to 10% adulteration in raw meat products. Discussion: This integrated approach leverages the complementary strengths of RPA and CRISPRCas12a: RPA enables rapid, sensitive amplification under isothermal conditions, while CRISPRCas12a ensures precise target recognition, effectively suppressing false-positive signals. Together, they form the foundation for a rapid, reliable, and field-applicable detection platform. The demonstrated performance highlights the assay’s strong potential as a robust, practical solution for on-site market surveillance, addressing the critical need for alternatives to complex, instrument-dependent laboratory methods. Conclusion: This study establishes a rapid, specific, and highly efficient RPA-CRISPR/Cas12a assay as a field-deployable tool for market surveillance agencies, enabling on-site screening of beef adulteration and supporting the integrity of food authenticity verification.