Research On The Identify Animal Medicinal Materials Of Chinese Pharmacopoeia By DNA Fingerprinting

Animal medicinal materials are important parts of traditional Chinese medicine resources.Compared with botanical materials,animal medicinal materials are characterized by multiple origin,and lack of specific secondary metabolites.It has been a difficult problem with the identification of traditional Chinese medicine because of the unclear origin and the lack of specific identification methods.As the development of molecular identification technology in recent years,various DNA identification methods have been applied to identify the authenticity of animal medicinal materials.Specific PCR technology and DNA barcode technology have been included in the Chinese Pharmacopoeia and have become legal testing bases.However,these DNA identification methods are difficult to guarantee both universality and specificity.In addition,those methods only focus on distinguish authentic from adulterants,and it is doesn’t concern how to identify admixture.Thus,the further promotion and application of them are restricted.Based on STR typing technology,a simple universal and specific method,universal DNA fingerprint,is established in this study for species identification of medicinal animal as well as admixture identification and quantitative analysis according to the characteristics of animal medicinal materials.The main research contents and results in this study are as follows:1.The Chinese Pharmacopoeia(2015 edition)contains animal medicines of 78 species from 14 classes.In this study,an overall collection of animal medicinal materials contained by Chinese Pharmacopoeia are conducted(including the original animal samples and the reference control medicinal materials of the National Institutes for Food and Drug Control)and 48 kinds of medicinal materials covering 59 species were collected.Samples of each species were replicated at least three times and were obtained up to 174 in total.To ensure the accuracy of their source,the samples were identified by morphology and confirmed by DNA sequence.The 57 complete mitochondrial genome sequence of animal medicinal materials,downloaded from GenBank,was analyzed in order to obtain the homologous genes by sequence alignment analysis.16 pairs of universal amplification primers were designed from the conserved sequences in both ends of the highly mutated region from the homologous gene.Amplification success rate and fluorescence STR typing success rate were tested.Finally,four pairs of universal primers that can obtain successfully the STR typing map of animal medicinal materials in the 2015 edition of Chinese Pharmacopoeia were selected.The selected primers were used for fluorescent STR typing of animal medicinal materials,and the DNA fingerprint data set of animal medicinal materials in the 2015 edition of Chinese Pharmacopoeia was obtained.Each species has a different DNA fingerprint,which can identify each other.2.Take bile powder,for instance,this paper expounds the application of the generic DNA fingerprint identification method of animal medicinal materials and adulterated medicines.The original animal or bile powders of pigs,cattle,sheep,chickens,ducks,geese,snakes,bears,fish were collected to extracted DNA.The fluorescein labeled primers,12S-L1091/12S-H1478 and 16S-L3428/16S-H3667,were sifted.The primers were used for PCR amplification,fluorescence TR typing and data analysis of the bile powder.Different species can be identified for their different STR fingerprints.Besides,the fingerprints have both the authentic and fake’s information,the adulteration of authentic and fake can be identified.The migration peak of STR typing map in mixed sample is co-dominant and can display multiple amplified fragments at the same time.We tried to identify the multiple mixed bile powder samples(pig,bovine sheep,chicken,duck,goose,snake,fish and bear)and found that DNA fingerprints can show the migration peaks of each species at the same time.3.Take the complicated and multiple sources medicinal pheretima,for instance,we tried to explore the potential for the generic DNA fingerprint identification method of animal medicinal materials for species identification with complex closely related species and quantitative(semi-quantitative)identification of admixture samples.The medicinal pheretima were collected(15 species,175 samples),the DNA were extracted.The generic DNA fingerprint identification primers12S-L1739/12S-H2175,12S-L1091/12S-H1478 and16S-L3428/16S-H3667 were sifted.The primers were used for fluorescence STR typing,data analysis to medicinal pheretima and the DNA fingerprint were obtained.The migration peaks 389.7,396.2,394.7,391.4 bp(12S-L1739/12S-H2175);410.9,416.7,415.9,411.8 bp(12S-L1091/12S-H1478);162.8,164.1,164.2,163.7 bp(16S-L3428/16S-H3667)were obtained respectively by using the three primers of Pheretima aspergillum,P.vulgaris,P.guillelmi,P.pectinifera.The migration peaks of 11 kinds of adulterants are different from authentic.DNA fingerprinting result formed by three universal primer could accurate characterization the species origin of pheretima.The authentic pheretima DNA and the adulterants pheretima DNA was mixed with 11 different concentration ratios,and the STR typing was performed by primers 12S-L1739/12S-H2175.The amplification products of the authentic pheretima DNA and the fake pheretima were ixed with the same concentration ratio and their STR typing result was employed as the reference result.The peak area ratio of the two experiments and mixed DNA concentration ratio of authentic and fake pheretima was compared.The results showed that the mixed reference ratio is closer to the mixed DNA concentration ratio.But the proportions were similar on the whole,which realized the quantitative identification of the adulteration of pheretima.In conclusion:The universal DNA fingerprinting method of the Chinese Pharmacopoeia for animal medicinal materials was preliminarily established in this research and the reference DNA fingerprint data set was obtained.The species identification,adulterant identification and quantitative adulterant identification of animal medicinal materials was realized.This method provides a new method of the identification of animal medicinal materials and lays a good foundation for ensuring the safety and effectiveness of clinical medication.