Identification of horse ingredients based on recombinase polymerase amplification, high-throughput DNA isolation and magnetic separation

Abstract

Adulteration of meat products remains a significant issue worldwide. In this study, we developed a high-throughput, rapid, sensitive, and selective method for qualitative and quantitative detection of horse meat adulteration in meat products. This method, which is not only innovative but also highly practical, involves coupling a 96-well high-throughput DNA isolation process with recombinant enzyme polymerase amplification (RPA), magnetic separation (MS), and high-throughput fluorescence detection (FD). We designed a set of primers targeting the mitochondrial ATP6-8 gene of horses. The upper and lower primers were labeled with 6-FAM fluorescent moiety and biotin, respectively. Following RPA amplification, the amplicons were separated and purified using streptavidin-modified magnetic beads (MB@SA). Subsequently, the amplicons were qualified and quantified using a high-throughput fluorescence detection system. We optimized the conditions for primer addition, incubation time, and the ratio of amplicon to MB@SA. Our results showed that a fluorescence threshold of 834 indicated positive detection. The method exhibited high specificity, with no positive amplification observed in DNA from the other 11 types of meat. It demonstrated high sensitivity and was capable of detecting as little as 0.1% (wt%) of horse meat in adulterated samples. Combined with our developed 96-well high-throughput DNA extraction system, the RPA-MS-FD assay process could analyze 96 samples in 31.5 min, including 11.5 min for rapid DNA extraction, 15 min for RPA amplification, and 5 min for fluorescence detection. Finally, we successfully applied this assay to analyze 21 commercial meat products, proving its practicality and effectiveness.