Abstract
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.
Highlights
Deinagkistrodon acutus (Günther, 1888), the sharp– snouted pit viper, is the only representative of the monotypic genus Deinagkistrodon
In the practice of law enforcement struggling against the illegal trade, officers often encounter lawbreakers who argue that what they are selling is not D. acutus but other snake species with similar body size and appearance, including related vipers such as Gloydius halys, G. intermedius, Ovophis monticola, Daboia siamensis, and even some rat snakes or elapid snakes with larger body size, such as Orthriophis moellendorffi, Euprepiophis mandarinus, Ptyas mucosus, and Naja atra (Su et al, 2016)
We established an recombinase polymerase amplification (RPA)–based method combined with fast DNA extraction for on–spot D. acutus identification that can be completed in 30 min at 37 oC
Summary
Deinagkistrodon acutus (Günther, 1888), the sharp– snouted pit viper, is the only representative of the monotypic genus Deinagkistrodon. In the practice of law enforcement struggling against the illegal trade, officers often encounter lawbreakers who argue that what they are selling is not D. acutus but other snake species with similar body size and appearance, including related vipers such as Gloydius halys, G. intermedius, Ovophis monticola, Daboia siamensis, and even some rat snakes or elapid snakes with larger body size, such as Orthriophis moellendorffi, Euprepiophis mandarinus, Ptyas mucosus, and Naja atra (Su et al, 2016). A recent article by Jiang et al (2015) described a homogeneous fluorescent specific PCR method using cationic conjugated polymer for the identification of medicinal snakes including D. acutus. All these molecular approaches for D. acutus identification do not allow efficient or convenient on–spot identification (Huang et al, 2014). We established an RPA–based method combined with fast DNA extraction for on–spot D. acutus identification that can be completed in 30 min at 37 oC
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