Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP

Abstract

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.