Development and validation of a two-step assay for differentiation of Penis et testis cervi from cervi nippon Temmink and cervi elaphus Linnaeus based on specific-species polymerase chain reaction-restriction fragment length polymorphism patterns

Abstract

Penis et testis cerviis one of the precious traditional Chinese medicines (TCMs) in China, which, whenauthentic, is sourcedfrom species ofCervi nippon Temmink(C. Nippon)andCervi elaphus Linnaeus(C.elaphus L).Authentication of species ofPenis et testis cerviremainstechnically challenging. This study attempted to modify the species-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method using restriction enzymeHpy188III for the identification ofPenis et testis cervispecies.First, thecytochrome b (Cytb)and cytochrome c oxidase subunit I (COI) genes in mitochondrial DNAfrom two authentic species and their counterfeitsor adulterantswere downloaded and analyzed with bioinformatics tools to establish the phylogenetic tree, then a series of primers were designed, and the restriction enzymesites were confirmed. Genomic DNA was extracted fromall samplesby the improved SDS method. Species-specific PCR reactions were optimizedto differentiate authentic Sika deer and ReddeerPenis et testis cervifrom their counterfeits or adulterants. PCR-RFLP using single restriction enzyme Hpy188III was performed inCytb regionsto identify the species of Sika deer and Reddeer. Simultaneously, recombination plasmid carrying thecytb fragmentcould be cloned, sequenced, and blasted.The assay specificity and sensitivity were evaluated, and30 batches of commercial samples werevalidated. Thephylogenetic treeshowed that the physico-chemical properties of Sika deer and Reddeer variants were similar.Sufficient DNA extracts were extracted from all samples used inthe improved SDS method.Establishment and optimization of a species-specific PCR reactionsand PCR-RFLPsystem were undertaken to identify deer species inPenis et testis cervisamples. Aspecific PCR-RFLP pattern with three distinct products of sizes 155, 113, and 204 bp was exhibited in Sika deerspecies,and two distinct products of 204 and 268 bp in size were evidentin Reddeerspecies,which were significantly different from the pattern of thecounterfeitsor adulterants.The distinctive PCR-RFLP fingerprint pattern using the restriction enzyme Hpy188III is a rapid and reliable assay for identification of thePenis et testis cervispecies.