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

Penis et testis cervi is one of the precious traditional Chinese medicines (TCMs) in China, which, when authentic, is sourced from species of Cervi nippon Temmink (C. Nippon) and Cervi elaphus Linnaeus (C. elaphus L). Authentication of species of Penis et testis cervi remains technically challenging. This study attempted to modify the species-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method using restriction enzyme Hpy188III for the identification of Penis et testis cervi species. First, the cytochrome b (Cyt b) and cytochrome c oxidase subunit I (COI) genes in mitochondrial DNA from two authentic species and their counterfeits or adulterants were downloaded and analyzed with bioinformatics tools to establish the phylogenetic tree, then a series of primers were designed, and the restriction enzyme sites were confirmed. Genomic DNA was extracted from all samples by the improved SDS method. Species-specific PCR reactions were optimized to differentiate authentic Sika deer and Red deer Penis et testis cervi from their counterfeits or adulterants. PCR-RFLP using single restriction enzyme Hpy188III was performed in Cyt b regions to identify the species of Sika deer and Red deer. Simultaneously, recombination plasmid carrying the cyt b fragment could be cloned, sequenced, and blasted. The assay specificity and sensitivity were evaluated, and 30 batches of commercial samples were validated. The phylogenetic tree showed that the physico-chemical properties of Sika deer and Red deer variants were similar. Sufficient DNA extracts were extracted from all samples used in the improved SDS method. Establishment and optimization of a species-specific PCR reactions and PCR-RFLP system were undertaken to identify deer species in Penis et testis cervi samples. A specific PCR-RFLP pattern with three distinct products of sizes 155, 113, and 204 bp was exhibited in Sika deer species, and two distinct products of 204 and 268 bp in size were evident in Red deer species, which were significantly different from the pattern of the counterfeits or adulterants. The distinctive PCR-RFLP fingerprint pattern using the restriction enzyme Hpy188III is a rapid and reliable assay for identification of the Penis et testis cervi species.