| DNA barcoding is a newly-developed molecular biotechnology, and now it is highlighted. In the Pharmacopioea of the People’s Republic of China (Chinese Pharmacopoeia for short) 2015 edition, DNA barcoding has been included, as a state standard. To make up for the deficiency of traditional identification methods (morphological identification, origin identification, microscopical identification, physiochemical identifation), DNA barcoding has been applied to the authentication of traditional Chinese medicine for more than ten years. In the present study, we take Acori Calami Rhizoma, Ilicis Chinensis Folium, Cuscutae Semen and Chinese patent medicine containing Ginseng Radix et Rhizoma, Panacis Quinquefolii Radix or Fritillariae Cirrhosae Bulbus as example, based on internal transcribed spacer 2 (ITS2), verify the variability of DNA barcoding. So that as efficient method for quality control and market supervision can be developed. Finally, conclsions are as follows:(1) Identification of Acori Calami Rhizoma for rhizomatic traditional Chinese medicine:Together with 49 internal transcribed spacer 2 (ITS2) sequences from GenBank,47 regions of Acorus calamus and related sequences were analysed. After sequcence alignment, phologenetic tree construction, single nucleotide polymorphism sites (SNP sites) detection, variable sites analysis and so on, two stable SNPs in ITS2 regionwere detected to identify Acorus calamus and its adulterants. The constructed Neighbour-joining tree can also identify A. calamus from the other species. Therefore, DNA barcoding together with SNPs could be used as an efficiently method to identify A. calamus and its adulterants.(2) Identification of Ilicis Chinensis Folium for leaves of traditional Chinese medicine:37 samples including Ilicis Chinensis Folium and its adulterants and related species were used to get genomic DNA for future sequencing. Accompanied by 28 ITS2 sequences from GenBank including Ilex genus, Ligustrum lucium, Symplocos setchuensis, interspecific and intraspecific diversity were analyzed, neighbor-joining tree were constructed. Finally Ilex chinensis can be identified in all the aspects. Therefore, DNA barcoding can efficiently differentiate I. chinensis from the morphologically similar species. As a fast, accurate, simple molecular biology, DNA barcoding can be used to identify Ilex chinensis and related species, and has a protential to identify species in Ilex genus.(3) Identification of Cuscutae Semen for Chinese semen medicine:A total of 207 ITS2 (Internal Transcribed Spacer 2) sequences representing forty-five related species of Cuscutae semen were collected to construct a standard DNA barcode database, then thirty-three commercial samples purchased from markets were analyzed by BLAST (Basic Local Alignment Search Tool) using the database, and Neighbor-joining tree was used to verify the efficacy of the database. The results revealed the percentage of counterfeits and adulterants in the thirty-three commercial samples was up to 69.7%, and only ten commercial products were found to be genuine. The adulterated species focused on eleven species (Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscu-ta japonica, Cuscuta monogyna, Foeniculum vulgare, Glycine max and Medicago sativa). The results showed the method developed in this study was quite efficient for Cuscutae semen identification and herbal market supervision.(4)Identification of Chinese patent medicine:Take Shedanchuanbei Capsule, Qipi Pill, Renshenjianpi Pill, Renshenguipi Pill, as example, we used next-generation sequence to detect constitutes of Chinese patent medicine based on ITS2 and short region (300 bp). Finally, we found that all these four medicines containing related species. What’s more, genuine Panax ginseng and related species Panax quinquefolium share the same ratio, which seriously threats the drug safety. |
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