A DNA Integrated System For Identifying Traditional Medicines

The authenticity of Chinese herbal medicine was the basis of its efficacy and safety. With the development of modern technology,conventional identification methods absorbed new technologies and methods constantly and promoted the development of modern identification methods. It is essential to establish a DNA integrated system for identifying traditional medicines in order to meet the needs of identification various Chinese herbal medicine.This study focuses on AAs-containing herbs and closely related species of Dioscorea genus, We used Taq Man probe and LAMP method to rapid detect Aristolochiaceae plants, DNA barcode to authenticate Aristolochiaceae plants and non-Aristolochiaceous plants, super barcode chloroplast genome to identify Dioscorea nipponica and its closely related species, Ultra High Performance Liquid Chromatography-Mass Spectrometer（UHPLC-HR-MS） to detect characteristic chemical constituent in Aristolochiaceous herbs,and on this basis we proposed the idea and the constituent part of a DNA integrated system for identifying traditional medicines,which laid a solid foundation in order to comprehensive identify wide variety of Chinese medicinal materials and solve some identification problems.We used Taq Man technology to rapid detect Aristolochiaceae plants.Aristolochic acids（AAs） is a class of organic compound wildly existed in the Aristolochiaceae plants which always widely used as Chinese traditional herbal medicines. However, for their similar Chinese name, pharmacy custom and plant morphology, the Aristolochiaceae plants were likely confused and misused with other medicinal materials. Aristolochic acid nephropathy incident caused by these confusion and misusing have got popularly attention. The traditional identification methods which mainly depend on the traits and microscopic features are difficult to be used to identify the dried plant materials and some medical material parts with less feature information. It is necessary to establish an effective method to identify the AAs containing plants to protect the consumers. In this study, we collected 289 samples, including 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families. We obtained 282 ITS2 sequences from 289 samples. Based on sequence information of ITS2, we developed a Real-Time PCR assay which successfully identified herbal material from the Aristolochiaceae family.We used LAMP technology to rapid identify Mu-tong（Akebiae Caulis）and its adulterant Guan-mu-tong（Aristolochiae Manshuriensis Caulis）. According to the Pharmacopoeia of the People’s Republic of China 2015, Mu-tong comes from the stems of Akebia quinata（Houtt.） Decne., Ak. trifoliata（Thunb.） Koidz. and Ak. trifoliata（Thunb.） Koidz. var. australis（Diels） T. Shimizu. A common adulterant of Mu-tong is Guan-mu-tong, which is derived from the stem of Aristolochia manshuriensis Komarov. We designed a set offour common primers（G-F3, G-B3, G-FIP, G-BIP） and a loop primer（G-LB） for LAMP based on ITS2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at an isothermal conditions of 65 ℃.Guan-mu-tong（Gmt-1 to Gmt-3） could be detected by LAMP within 25-30 min and saturated at 45-60 min. In contrast, LAMP of the three Akebia species（Mt-1 to Mt-9） showed negative until 60 min into the reaction. When the LAMP reaction completes, positive reactions（Gmt-1 to Gmt-3） changed from orange to green, whereas the negative reactions（Mt-1 to Mt-9） remained orange. The LAMP reaction exhibited a tenfold increase in detection over conventional PCR demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterants Guan-mu-tong.DNA barcoding technology was been used to identify Aristolochiaceae plants and its adulterants. We collected 289 samples, including 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families. In addition, 15 ITS2 sequences from 13 Asarum species, two ITS2 sequences of Pteroxygonum giraldii, four ITS2 and four psb A-trn H sequences of Hemsleya graciliflora were downloaded from Gen Bank.ITS2 was successfully amplified from 282 samples and successful amplification of psb A-trn H was obtained from 185 plant samples, and sequences were submitted to Gen Bank. We analyzed those sequences using DNA barcodes based on the ITS2 and psb A-trn H sequences. The results showed that the ITS2 and psb A-trn H sequences could both beused to successfully identify sample differences at the genus level（100% success rate） when BLAST1 and the nearest distance methods were used. The NJ tree showed that samples from the Aristolochiaceae family cluster together, whereas non-Aristolochiaceous samples clustered into their own clades. Therefore, the ITS2 as the core and psb A-trn H as supplementary DNA barcodes can be successfully used to distinguish Aristolochiaceous species from their putative substitutes（non-Aristolochiaceae）.Super barcode chloroplast genome has been applied in identification closely related species that DNA barcoding cannot give a solution and in this study we take identification Dioscorea nipponica and its closely related species as case. Dioscorea nipponica is an important medicinal plant belonging to Dioscoreaceae, a family is vital for evolution of monocotyledon.In the previous study, there was no appropriate DNA barcode for identification Dioscorea species. In the present study, the sequence of the D. nipponica chloroplast genome was determined. It was sequenced using Illumina Miseq Genome Analyzer with pair-end300 reads, and then assembled by Soapdenovo suite. The annotation is performed according to the preciously described method with default parameters. The chloroplast genome of D. elephantipes（NC₀09601.1） was served as reference. The assembling sequence has been deposited into Gen Bank with the accession number KP404629. It was an AT-rich（63.3%） chloroplast genome with 152,946 bp in length,containing a pair of 23,113 bp inverted repeats, which were separated by a large and a small single copy region of 83,557 bp and 23,064 bp in length, respectively. It encodes 120 unique genes,including 89 protein coding genes, 27 t RNA genes and 4 r RNA genes.The predicted gene coding regions covered 58.7% of the genome sequences. Ten genes contained one intron, while two genes had two introns. Phylogenetic analyses showed the present chloroplast genome can be used as a supper barcode to distinguish D. nipponica from its closely related species. Furthermore, the chloroplast genome provides us a molecular base for next investigation on this important medicinal species.UHPLC-HR-MS was applied in detection characteristic chemical constituent, and in this study we take detection AAⅠ and Ⅱ in Aristolochiaceous herbs as case. We collected a broad range of materials, 45 species from Aristolochiaceae family and 30 species of non-Aristolochiaceae family were detected by UHPLC-HR-MS. In this study, AAⅠ and Ⅱ were determined at m/z 340.0469 and310.0366, respectively. For Aristolochiaceous herbs, the results showed that 39 species contained AAⅠand 14 species also contained AAⅡ. Our results confirmed that plants from non-Aristolochiaceae family did not contain AAⅠ or Ⅱ. We demonstrated that most representatives from the Aristolochiaceae family contain AAs, thus UHPLC-HR-MS technology provide verified and complementary method to identify species contained aristolochic acid.Modern science and technology has shown great advantages in identification of medicinal plant, so our greatest concern is how to choose appropriate identification methods according to the different identification needs, in the face of various new methods and new technology. This research established a DNA integrated system for identifying traditional medicines, proposed the constituent part and identification process, and elaborated the every process of the DNA integrated system for identifyingtraditional medicines, which provided the basis for choosing appropriate identification method and laid a solid foundation in order to control the source and quality of the Chinese medicinal materials.A DNA integrated system for identifying traditional medicines includes the rapid detection system with Taq Man probes and LAMP method, DNA barcode system, super barcode chloroplast genome system for identification closely related species. The identification process of the DNA integrated system for identifying traditional medicines is divided into four major sections from a perspective of applied study. Firstly, in order to identification species in rapidly, sensitively and easily, we used the rapid detection system for rapid authentication. Secondly, in order to construct a DNA barcode libary or for species identification and traceability, DNA barcodes were used to obtain the sequence and identify species.Thirdly, super barcode chloroplast genome is used to identify closely related species when DNA barcodes cannot give a solution for identification closely related species. Lastly, for normal sample with extracting DNA easily, LC or LC-MS detection is the validation of DNA identification method; for the sample of DNA degradation or difficult to obtain DNA, LC or LC-MS detection is the effective complement of DNA identification method.