Novel CRISPR/Cas12-based assay for the rapid and accurate detection of donkey meat.

Background: Precise identification of items originating from animals is required for monitoring the authenticity of livestock commodities. Nucleic acid-based detection methods, including polymerase chain reaction, are highly accurate tests for detecting meat fraud. However, these tests require costly devices and highly skilled personnel. Aim: This study aims to develop a precise and rapid test based on deoxyribonucleic acid (DNA) for detecting meat from donkeys. Methods: The assay was developed by combining Clustered regularly interspaced short palindromic repeat /Cas12 with RPA and a lateral flow device. A conserved sequence of the mitochondrial D-loop gene was used as the target gene. The assessment of the assay focused on its sensitivity, specificity, and completion time. Results: The results show that the assay can detect donkey meat in the tested samples with 100% specificity with no cross-reactivity with other species, and no false-positive results were recorded. Furthermore, the assay has shown high sensitivity and to detect as little as 5 ng of input DNA. Moreover, the results, including DNA extraction, were obtained in less than 40 minutes and did not require any specialized equipment. Conclusion: The assay is a good option for the rapid and precise detection of donkey meat. Further optimization and studies are needed to enable the direct detection of donkey meat without DNA extraction.