Design and Validation of Specific Primers for COI Gene of Whitmania pigra Whitman
Objective To establish a polymerase chain reaction(PCR)method for rapid identification of Whitmania pigra Whitman and its common counterfeits.Methods A set of specific primers were designed for the identification of Whitmania pigra Whitman,based on the sequence of cytochrome c oxidase subunit Ⅰ(COI)and single nucleotide polymorphism(SNP)mutation sites.The stability,specificity and sensitivity of the primers were confirmed,and the application of the primers in Chinese patent medicine containing leech was verified.Results The specific primers named KT 1-3 or KT 1-4 were used to amplify the deoxyribonucleic acid(DNA)of leech,and the products were 235 bp and 266 bp,respectively.The LOD of this method was 0.0 1ng.The specific primer pair can be used to detect Whitmania pigra Whitman in simulated preparations of Naoxueshuan tablet and some commercial Naoxueshuan tablets.Conclusion The primers of the DNA molecular identification method has high specificity and sensitivity,which can be used as a supplement to the traditional identification method of leech medicinal materials and provide support for the rapid identification of Whitmaniapigra Whitman.
DNA barcodeWhitmania pigra Whitmanspecies-specific primersadulterationorigin identification