Recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a threatened species

This study addresses the use of recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a snake species threatened due to over–exploitation and habitat destruction. For its conservation, an efficient species identification method is urgently neededto fight against illegal capture and trade. Fourteen individuals representing 12 snake species (including D. acutus and other snake species) were collected from mountainous regions in Southern China. Genomic DNA was extracted within five minutes by a modified alkaline lysis method. Species–specific primers for recombinase polymerase amplification (RPA) were designed based on the sequences of cytochrome C oxidase subunit I (COI) barcode region, and an optimized RPA assay system was set up. Specificity and sensitivity of the assay were checked, and the assay was validated by identifying 10 commercial Qi She crude drug samples derived from D. acutus. Under optimized RPA conditions, a distinct single band of 354 bp was amplified only for D. acutus but not for the related snake species. The entire procedure can be completed in 30 min at room temperature. Commercial Qi She crude drug identification validated effectiveness of the established assay system. Using a recombinase polymerase amplification (RPA) assay with rapid DNA extraction, we established an on–spot D. acutus identification method with good specificity and sensitivity. This method could become an efficient tool for rigorous supervision of illegal D. acutus capture and trade.