The Molecular Detection Of Deer Products Based On Mitochondrial DNA Sequence

In our country, most deer products are rare traditional Chinese medicines, and products from sika deer (Cervus nippon) and wapiti (Cervus canadensis) are considered to be genuine, as described in old medicinal books and modern pharmacopeias for traditional Chinese medicine. Deer products are expensive, and merchants usually substitute them with inferior products to obtain lots of economic profits, so it is very significant to detect deer products. At present, detection methods of deer products based on mitochondrial DNA sequences mainly include PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), Sequence Analysis and Species-specific PCR. These methods can be effectively applied to the detection of deer products, however, the use of these methods seems to be complex and time-consuming due to many animal-derived ingredients used to substitute deer products. Therefore, it is necessary to design a simpler and faster method.Firstly, we bettered a method for isolating genomic DNA from mammalian whole blood and modified the traditional phenol-chloroform method. The former has many advantages contain non-toxic agents, low cost, high yield, simple operation and the whole process less than one hour, and the latter more fits to the extraction of genomic DNA of trace deer products.Secondly, we designed primers for amplifying the complete mitochondrial genome of Cervidae by using complete mitochondrial genomic DNA sequences of Cervidae in the GenBank database, and used these primers to sequence the complete mitochondrial genomic DNA from Northeast sika deer (Cervus nippon hortulorum), Northeast wapiti (Cervus elaphus xanthopygus) and Tarim red deer (Cervus elaphus yarkandensis), then submitted these sequences, had been noted and analyzed, to the GenBank database. After aligning these sequences in this paper and complete mitochondrial genomic DNA sequences of Cervidae in the GenBank database by Clustal X 1.83 software, we analyzed phylogenetic relationship about sika deer (Cervus nippon), wapiti (Cervus canadensis,Eastern lineage of Cervus elaphus) and red deer (Cervus elaphus,Western lineage of Cervus elaphus) by MEGA 4.0 software. The results show that: Sika deer contains three distinct branches, namely, Chinese population (mainland and Taiwan), southern population and northern population of Japan. The ancestor of Japanese sika deer migrated from the Asian mainland through at least two continent bridges in the late Pleistocene, then differentiated into southern population and northern population, and southern population had a closer genetic relationship with the mainland sika deer. In addition, Formosan sika deer (Cervus nippon taiouanus) could have evolved to the species level. Cervus elaphus originated in Central Asia, then differentiated into two different species, namely, wapiti (Asia-North American population) and red deer (European population) by migrating along two different directions, i. e. the east and the west. Wapiti has a closer genetic relationship with sika deer, and Tarim red deer is the ancestor of red deer. Sika deer and wapiti had latest common ancestor, with the ancestor of red deer, migrated along two different directions respectively, and differentiated eastward into sika deer and wapiti, westward into red deer in the end.Finally, after aligning complete mitochondrial DNA sequences in this paper and complete mitochondrial genomic DNA sequences of Cervidae, porcine (Sus scrofa), bovine (Bos taurus) and ovine (Ovis aries) in the GenBank database by Bioedit 7.0.9.0 software, we designed species-specific primers, including sika deer, wapiti, Tarim red deer, red deer, reindeer (Rangifer tarandus) and bovine, by Oligo 6.0 software based on appropriate regions selected according to the principle that these primers must have intraspecies-universality and interspecies-specificity. Then combined with porcine, ovine and poultry-specific primers in the published papers, we built up two sets of Multiplex PCR methods, which could detect various types of deer products in the positive side and the negative side, and both of these proved to be stable, reliable, fast and low-cost. Besides, we designed sambar-specific primers, and then established sambar-specific PCR to detect products from sambar (Cervus unicolor).