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

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.

This study aims to develop a precise and rapid test based on deoxyribonucleic acid (DNA) for detecting meat from donkeys.

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.

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.

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.