| In recent five editions of Chinese Pharmacopoeia,the proportion of multi-original medicinal materials has gradually increased and the number of Chinese patent medicines and prescription of traditional Chinese medicine involved in multi-original medicinal materials is huge,but the pharmacological activities,effective ingredient and content of medicinal materials of different originals are not exactly the same,which leads to differences in the quality of the materials.Multi-original medicinal species are mostly close related,with little difference in morphological characteristics.Using single-gene sequences to identify multi-original medicinal species has low efficiency,and even some genotype sequences were exactly the same.Due to the lack of natural resources and the difficulty of artificial cultivation,the precious Chinese medicinal materials are in short supply,resulting in fake or inferior products of Chinese medicinal materials,which seriously affects the safety of clinical medicine and the efficacy of Chinese medicine.Rhubarb is a medicinal rhubarb of Polygonaceae family,dried rhizomes of R.officinale,R.palmatum and R.tanguticum.It is an important traditional Chinese traditional medicinal material and is a typical multi-original medicinal material.It has the effects of alleviating stasis,clearing heat and cooling blood,activating blood circulation,dredging collaterals and stabilizing viscera,and is used to treat constipation of real heat accumulation,blood heat vomiting,red eye pharyngeal swelling,carbuncle swollen boils,intestinal carbuncle abdominal pain,blood stasis and amenorrhea,postpartum stasis,bruises,damp heat diarrhea,jaundice urinary red.The medicinal materials of rhubarb plants from different species will inevitably lead to differences in their pharmacological activity and efficacy due to differences in active ingredients and pharmacological activities.In order to quickly and accurately determine the three species and ensure accurate drug application,first obtain one fresh plant from the three authentic production area of three species,extract chloroplast genomic DNA,sequence using next-generation sequencing technology,and analyze the chloroplast genome of the three species through gene assembly and annotation.R.officinale chloroplast genome size is161,093 bp,R.palmatum chloroplast genome size is 161,541 bp and R.tanguticum chloroplast genome size is 161,053 bp.A total of 131 functional genes were annotated in three species,including 91 protein-coding sequences,8 r RNAs,and 31 t RNAs,of which 7 genes contained 1 intron,2 genes contained 2 introns,and 18 genes repetitive 2 copies,3 copies of 1 gene.Analyze the number and type of simple sequence repeats(SSR)in the chloroplast genomes of three species.The mono-nucleotide have the largest proportion;the number of SSR were different in large single copy region(LSC),inverted repeats(IRA and IRB)and small single copy region(SSC).The length of most SSR <20bp.Using the chloroplast genomes of 23 species including three rhubarb-based species to build an evolutionary tree,the three rhubarb-based species preferentially gather into one branch.Based on the differences in the chloroplast genomes information of the three species of Rhubarb,21 pairs of specific primers were designed for the regions with large differences in the chloroplast genomes of the three species to screen candidate sequences that can accurately and quickly identify the three species.After PCR amplification,sequencing of amplified products and comparison of amplified sequence information,the amplified fragments of 8 pairs of primers were screened out.Through base substitution,base deletion and base insertion,accurate identification of the three basic source species of rhubarb medicinal materialsB.multicinctus is a traditional Chinese medicine with significant pharmacological activity.It has the functions of dispelling wind and removing obstruction in the meridians.It is used to treat rheumatic arthralgia,numbness and convulsions,stroke,slanting eyes and mouth,hemiplegia,convulsions and spasms,tetanus,leprosy and scabies.Due to the outstanding curative effect of this medicinal material in the treatment of rheumatoid arthritis,epilepsy,urticaria and cervical spondylosis,leading to a large clinical demand,resulting in the counterfeit of this variety of medicinal materials frequently appear in the Chinese medicine market.Design specific primers for the COI sequences of mitochondrial genes about B.multicinctus and common fake,and using MLPA to detect B.multicinctus,S.annularis,X.flavipunctatus,D.acutus and N.atra were identified by simultaneous identification method and the amount of authentic and counterfeit samples in the mixed sample was analyzed based on the relative peak area of the capillary electrophoresis signal peak.The MLPA method was used to identify five species.The single probe and the probemixhad strong specificity and there was no mutual interference between the species.This method showed strong specificity.When the proportion of sample DNA in the mixed sample was greater than 30%,the content of the mixed sample can be inferred based on the peak area of the capillary electrophoresis.When the proportion of sample DNA in the mixed sample was less than 30%,it can be used to identify whether adulteration exists in the mixed sample.In summary,through the chloroplast whole genome to realize the rapid identification of the three original species of rhubarb,the MLPA method can realize the simultaneous identification and relative quantitative analysis of the B.multicinctus and its common counterfeit.The accurate,rapid and standardized identification of the two varieties Chinese medicinal material at the molecular level by the two methods can verify the effectiveness,stabilityand scientificity of the two methods.These two methods can be extended to the identification of other multi-original medicinal materials and valuable medicinal materials. |
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