中藥鑒定創(chuàng)新方法體系的建立與推廣課件

1、中藥鑒定創(chuàng)新方法體系的建立與推廣中藥鑒定創(chuàng)新方法體系的建立與推廣中藥鑒定技術(shù)瓶頸 一萬余種中藥材物種鑒定 品種混亂傳統(tǒng)四大鑒定方法體系的局限 路在何方？基原鑒定形態(tài)鑒定顯微鑒定理化鑒定中藥鑒定技術(shù)瓶頸 一萬余種中藥材物種鑒定 品種混亂DNA條形碼 中藥鑒定領(lǐng)域的變革與突破經(jīng)典中藥鑒定方法難以滿足現(xiàn)代需求鑒定方法缺失導(dǎo)致嚴(yán)重安全性事件 - 關(guān)木通誤當(dāng)木通服用，導(dǎo)致急性腎功能衰竭（馬兜鈴酸腎病事件） -土三七誤當(dāng)三七服用，導(dǎo)致嚴(yán)重肝毒性Hoang et al. Science Translational Medicine, 2013, 5: 197ra102.Nortier J L, Martin

2、ez M C M, Schmeiser H H, et al. New England Journal of Medicine, 2000, 342(23): 1686-1692.關(guān)木通百度百科木通中藥材天地網(wǎng)DNA條形碼 中藥鑒定領(lǐng)域的變革與突破經(jīng)典中藥鑒定方法難以DNA barcoding can achieve a rapid and accurate identification for herbsA powerful tool for identifying medicinal plants and traditional Chinese medicinal materialsAbr

3、us cantoniensis Abrus precatorius Medicinal plantsChinese herbal medicinesDNA barcoding technology uses a short and standardized gene region to identify speciesOne sequence One speciesDNA barcoding can achieve a ra課題組首次提出并驗證ITS2條形碼，引起國內(nèi)外廣泛關(guān)注 國際期刊PLoS ONE關(guān)注藥用植物DNA條形碼。該文提出ITS2序列為藥用植物通用的條形碼序列，為陸地植物DNA條

4、形碼序列篩選提出了新的視角，由此引起同行的關(guān)注和討論。被德國專業(yè)網(wǎng)站選為近25年來在ITS2領(lǐng)域最受關(guān)注的16篇文章之一http:/its2.bioapps.biozentrum.uni-wuerzburg.de/該文發(fā)表后3年引文288次課題組首次提出并驗證ITS2條形碼，引起國內(nèi)外廣泛關(guān)注 首次驗證 ITS2為藥用植物鑒定通用條形碼通過研究比較psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2 和 ITS等七個候選DNA條形碼，發(fā)現(xiàn)ITS2是最佳條形碼序列ITS2長度約230bp，易于擴增和測序，物種鑒定能力強，適合中藥材等存在DNA降解材料的鑒定人參、西洋參

5、、羌活、寬葉羌活DNA條形碼鑒定方法正在納入國家藥典首次驗證 ITS2為藥用植物鑒定通用條形碼通過研究比較psb Relative distribution of inter-specific divergence between congeneric species and intra-specific variation for four loci. The two bars in each box represent inter-specific (above) and intra-specific (below) genetic distances.Identification of

6、the family RosaceaeW+W-Relative Ranks, n, P valueResultITS2matKW+ = 703, W- = 0, n = 37, P 1.139 10-7ITS2 matKITS2rbcLW+ = 703, W- = 0, n = 37, P 1.137 10-7ITS2 rbcLITS2rpoC1W+ = 703, W- = 0, n = 37, P 1.133 10-7ITS2 rpoC1matKrbcLW+ = 620, W- = 46, n = 36, P 6.479 10-6matK rbcLmatKrpoC1W+ = 655, W-

7、= 11, n = 36, P 4.174 10-7matK rpoC1rbcLrpoC1W+ = 359, W- = 106, n = 30, P 0.009rbcL rpoC1Wilcoxon signed-rank tests of inter-specific divergence among loci.ITS2 and rbcL, matK, rpoC1 were compared.For 54 samples from 43 species in 19 diverse genera, the successful identification rate with ITS2 was

8、100%, followed by matK (94%) and rbcL(65%). For 1410 samples from 893 species in 96 genera No. of genera (2 species): 47 (1351 samples) No. of species ( 2 samples ): 297 (814 samples) Identification rate with ITS2 was 100% and 78% at genus and species level, respectively.X Pang, Shilin Chen*. Cladis

9、tics, 2011, 27 Relative distribution of inteComparative analysis of a large dataset indicates that ITS should be incorporated into the core barcode for seed plantsChina Plant BOL Group, Li Dezhu et al., PNAS, 2011 We therefore propose that ITS/ITS2 should be incorporated into the core barcode for se

10、ed plants. (建議ITS/ITS2應(yīng)成為種子植物的核心條形碼)In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful back-up as it is easier to amplify and sequence this subset of the marker. (ITS難以擴增和測序時，ITS2可以有效地彌補該缺陷)Comparative analysis of a larg創(chuàng)建ITS2為主體條形碼的中草藥鑒定

11、體系研究一萬余份藥材樣本（涉及數(shù)千個基原物種）70余篇相關(guān)文章發(fā)表于Cladistics、PLoS ONE、PNAS、BMC Evol Biol、 中國科學(xué)等雜志出版中藥DNA條形碼分子鑒定 人民衛(wèi)生出版社起草中國藥典中藥材DNA條形碼分子鑒定指導(dǎo)原則，已獲準(zhǔn)納入國家藥典建立中藥DNA條形碼分子鑒定標(biāo)準(zhǔn)流程創(chuàng)建ITS2為主體條形碼的中草藥鑒定體系研究一萬余份藥材樣冬蟲夏草混偽品及近緣種人造偽品：滇紫菀冬蟲夏草及其混偽品/近緣種背景：分布范圍狹窄，生態(tài)環(huán)境脆弱，資源蘊藏量有限（產(chǎn)量每年80-100噸）； 市場產(chǎn)不足銷，價格已超黃金 (Guo, 2012) 混偽品泛濫，如古尼蟲草，蛹蟲草，人造偽品

12、滇紫菀等 樣品：冬蟲夏草與13種混偽品及近緣種，冬蟲夏草采自四川、青海、西藏等主產(chǎn)區(qū) 185 條ITS/ITS2序列 (冬蟲夏草樣品53份，偽品45份，GenBank序列87 條)A：正品：冬蟲夏草B：偽品： 亞香棒蟲草/古尼蟲草C：人造偽品： 滇紫菀D：偽品 涼山蟲草E：偽品：蟬花F：偽品：下垂蟲草G：偽品： Cordyceps robertsii（新西蘭） 結(jié)果：基于BLAST/DISTANCE的鑒定效率：100% 基于NJ tree 的鑒定結(jié)果：所有的冬蟲夏草序列單獨聚為一支， 并能與所有的混偽品及近緣種區(qū)分開 Xiang L, Chen SL, et al. FEMS microbio

13、logy letters, 2013, 347(2): 156-162.DNA條形碼鑒定冬蟲夏草及其偽品冬蟲夏草混偽品及近緣種人造偽品：滇紫菀冬蟲夏草及其混偽品/近中國藥典中藥材DNA條形碼分子鑒定指導(dǎo)原則中國藥典中藥材DNA條形碼分子鑒定指導(dǎo)原則Morphological characteristics of the powder, decoction pieces, seed ,seedling, and fibre among the P.ginseng , P. quinquefolius, P notoginseng and its closely related species a

14、re similar.It is difficult to identify so many different panax species and ginseng produce in medicinal uses by traditional classification methods.Using DNA barcode to identify panax medicinal species 人參類及其混偽品的鑒定Morphological characteristics13 1 5 10 15 20 25 30 35 40 45 50SN01: p.ginseng;SN03: p.qu

15、inquefolius 顯示人參、西洋參ITS2序列的SNPs位點信息。Position 32 and 43為區(qū)別人參和西洋參的SNPs位點為驗證該方法準(zhǔn)確性，我們下載了GenBank所有人參、西洋參ITS2序列，表明兩個SNPs位點穩(wěn)定存在樣品來源：采集了主產(chǎn)區(qū)吉林，遼寧，黑龍江，北京，山東,加拿大及上海和香港藥市人參西洋參樣品共計72份應(yīng)用ITS2序列兩個SNPs可以快速準(zhǔn)確鑒別人參西洋參，所得組合為C-T，則確定為人參，所得組合為T-C，則確定為西洋參ITS2 sequence position 1-50 of p.ginseng and p. quinquefolius 方 法結(jié) 果結(jié)

16、 論基于ITS2條形碼鑒定人參和西洋參Gene, 2013, 530(1): 39-43.A Fast SNP Identification of Medicinal Panax Species Based on DNA Barcoding Analysis, 15 1 5 14金銀花藥材及其混偽品的ITS2條形碼鑒定金銀花（忍冬）山銀花（紅腺忍冬）山銀花（黃褐毛忍冬）山銀花（灰氈毛忍冬）研究對象：包括24份金銀花藥材。M: marker1、26: 陰性對照(CK) 2-25: 金銀花27-47：混偽品DNA提?。喝〗疸y花藥材約25 mg，利用植物基因組DNA提取試劑盒(Tiangen Bio

17、tech Co., China)提取總DNA。ITS2序列PCR擴增程序及電泳圖94 變性5 min94 變性30 sec58 退火30 sec72 延伸45 sec72 延伸10 min40個循環(huán)BLAST鑒定16金銀花藥材及其混偽品的ITS2條形碼鑒定金銀花（忍冬）山 Identification of Cortex HerbsSun ZY, Chen SL*. J Nat Med. 2012.The identification efficiency of 51 samples belonging to 19 kinds of cortex herbs in Chinese Pharma

18、copoeia examined was 100% using ITS2 sequence as DNA barcode.Apply the ITS2 barcode to authenticate medicinal materials 皮類藥材的條形碼鑒定 Identification of Cortex Herb條羌N. franchetii大頭羌N. franchetii蠶羌N. incisumPhylogenetic tree constructed with the ITS/ITS2 sequencesadulterantsITSITS231 medicinal materials

19、 samplesStability and Accuracy of the Identification using ITS/ITS2 barcodes (Notopterygii )羌活藥材ITS/ITS2條形碼鑒定及其穩(wěn)定性與準(zhǔn)確性研究The NJ trees showed that Qianghuo and its adulterants can be easily differentiated according to their monophyly.ITSITS2HaplotypeVariable sitesHaplotypeVariable sitesN. incisum4433N

20、. franchetii61045Xin T Chen S *, et al. Acta Pharm Sin. 2012, 8條羌N. franchetii大頭羌N. franchetiUse of the potential DNA barcode ITS2 to identify herbal materials 全草類DNA條形碼鑒定 The ITS2 barcode could distinguish successfully all the 34 herbs from their 111 adulterants.Figure 1. The presence/absence of ba

21、rcode gaps between herbs and their adulterants. Pang XH, Chen SL* et al. J Nat Med. 2012Use of the potential DNA barco建立中藥材DNA條形碼鑒定系統(tǒng)網(wǎng)站建立中藥材DNA條形碼鑒定系統(tǒng)網(wǎng)站http:/www.tc DNA barcode system shows strong abilities in identifying herb cryptic species BLAST PROCESS打開網(wǎng)址，進(jìn)入中藥材DNA條形碼鑒定系統(tǒng)主頁點擊物種鑒定，選擇植物類藥材鑒定（ITS2/

22、ITS）進(jìn)入右圖界面。 DNA barcode system shows 在空白處粘貼入需要校對的序列（Query）點擊左下角的提交比對序列相似性信息100.0%為人參，說明該序列鑒定結(jié)果為人參Quick-and-easy DNA identification of herbs is under way在空白處粘貼入需要校對的序列（Query）點擊左下角的提交比華潤三九中藥材DNA條形碼鑒定專用軟件華潤三九中藥材DNA條形碼鑒定專用軟件2022/9/92022/9/5 七葉皂苷注射劑娑羅子藥材的基原植物為七葉樹Aesculus chinensis、浙江七葉樹Aesculus chinensis

23、 var. chekiangensis和天師栗Aesculus wilsonii娑羅子藥材DNA條形碼鑒定1. 材料 七葉樹屬10個物種42份樣本采自云南、四川、陜西、湖北、浙江和北京的各個分布區(qū)域結(jié) 論七葉樹屬候列擴增效率與測序成功率均為100%，并且物種鑒定效率最高，為95.4%，更適用于七葉樹屬的鑒定分析。psbA-trnH序列的種間差異最大，在區(qū)分物種上具有優(yōu)勢?；趐sbA-trnH序列構(gòu)建的NJ樹，可準(zhǔn)確鑒別其各個基原物種。七葉樹屬植物psbA-trnH序列比對變異位點圖正品 七葉皂苷注射劑娑羅子藥材的基原植物為七葉樹Aesc柴胡 Chaihu正品：為傘形科植物柴胡Bupleuru

24、m chinense DC.、狹葉柴胡B. scorzonerifolium Willd.的干燥根。近緣種：同屬植物麗江柴胡B. rockii，小葉黑柴胡B. smithii var. parvifolium ，抱莖柴胡B. longicaule var. amplexicaule，細(xì)莖有柄柴胡B. petiolulatum var. tenerum 和窄竹葉柴胡B. marginatum var. stenophyllum。（三）柴胡與其近緣種的NJ樹圖所示：柴胡正品基原柴胡與狹葉柴胡均獨立聚為一支，與其混偽品可以區(qū)別開。結(jié)果表明：基于ITS2序列能很好的區(qū)分柴胡及其近緣種（一）柴胡及其及其

25、近緣物種ITS2序列的二級結(jié)構(gòu)：（二）柴胡與其混偽品ITS2序列間存在較多變異位點柴胡不同基原ITS2 條形碼consensus 序列柴胡ITS2序列峰圖柴胡 Chaihu正品：為傘形科植物柴胡Bupleurum 常用中藥材進(jìn)行了DNA條形碼研究課題組已經(jīng)進(jìn)行DNA條形碼研究的常用中藥材包括：人參，西洋參，三七，大黃，石斛，重樓，金銀花，丹參，天南星，紅豆蔻，肉蓯蓉，京大戟，麻黃，高良姜 ，何首烏，吳茱萸，連翹，甘草，玉竹，冬蟲夏草，羌活等常用中藥材進(jìn)行了DNA條形碼研究課題組已經(jīng)進(jìn)行DNA條形碼研 Published papers on DNA barcode Cladistics, 2011, 27:BMC Evol. Biol., 2010, 10:324 PLoS ONE, 2010,5: e13102.China Plant BOL Group. PNAS, 2011,108(49) FEMS Microbiology Letters, 2013 PLoS ONE, 2012, 7(5):e35146. Jingyuan Song, Shilin Chen*. PLoS ONE, 201