DNA宏条形码及其在混合中草药物种鉴定中的应用
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摘要
DNA宏条形码(DNA metabarcording)是利用高通量测序技术获得混合条形码扩增序列,通过生物信息学分析手段来鉴定混合样本中物种组成的方法,已广泛用于宏基因组学、动植物生物多样性等研究,并开始应用于传统草药的鉴定领域,为混合草药物种鉴定困难提供了有力的手段。该文主要总结了目前DNA宏条形码在混合中草药基原物种鉴定研究中的应用进展,重点比较分析了Sanger一代、二代以及三代高通量测序技术在混合中草药鉴定中的优劣性,并展望DNA宏条形码在混合中草药产品鉴定中的应用前景。
DNA metabarcoding,one rapid and robust method using specific standard DNA fragments,has been widely used for rapid species identification of a bulk sample through high-throughput sequencing technologies.While it has been widely used in the studies of metagenomics,animal and plant biodiversity,it has gradually come to be used as a profitable method in species identification of mixed Chinese herbal medicines.In this paper,we mainly summarize the current studies of the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines.Moreover,high-throughput sequencing technologies adopted in those studies,such as Sanger,the next-generation,and third-generation sequencing technologies,are discussed.It is conducted to provide a theoretical guidance for the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines and in more other biodiversity studies.
DNA metabarcoding,one rapid and robust method using specific standard DNA fragments,has been widely used for rapid species identification of a bulk sample through high-throughput sequencing technologies.While it has been widely used in the studies of metagenomics,animal and plant biodiversity,it has gradually come to be used as a profitable method in species identification of mixed Chinese herbal medicines.In this paper,we mainly summarize the current studies of the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines.Moreover,high-throughput sequencing technologies adopted in those studies,such as Sanger,the next-generation,and third-generation sequencing technologies,are discussed.It is conducted to provide a theoretical guidance for the application of DNA metabarcoding in species identification of mixed Chinese herbal medicines and in more other biodiversity studies.
引文
[1]Vlietinck A,Pieters L,Apers S.Legal requirements for the quality of herbal substances and herbal preparations for the manufacturing of herbal medicinal products in the European Union[J].Planta Med,2009,75(7):683.
[2]EMA.Guideline on the assessment of clinical safety and efficacy in the preparation of European Union herbal monographs for wellestablished and traditional herbal medicinal products EMA/HMPC/104613/2005 Rev.1[EB/OL].(2016-07-12)[2018-04-24].http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/09/WC500212240.pdf.
[3]李浩,田侃,喻小勇,等.欧盟成员国植物药产品审评现状分析及启示[J].中草药,2016,47(24):4494.
[4]崔伟锋,王素花,王会丽,等.真实世界中医临床研究方法与实践[J].中国全科医学,2016,19(13):1586.
[5]钟丽丹.中成药上市后再评价的现状、挑战与展望[J].世界中医药,2017,12(6):1218.
[6]Hebert P D N,Cywinska A,Ball S L,et al.Biological identifications through DNA barcodes[J].P Roy Soc Lond B Bio,2003,270(1512):313.
[7]Li X W,Yang Y,Henry R J,et al.Plant DNA barcoding:from gene to genome[J].Biol Rev,2015,90(1):157.
[8]Chen S L,Pang X H,Song J Y,et al.A renaissance in herbal medicine identification:from morphology to DNA[J].Biotechnol Adv,2014,32(7):1237.
[9]陈士林,姚辉,韩建萍,等.中药材DNA条形码分子鉴定指导原则[J].中国中药杂志,2013,38(2):141.
[10]Shendure J,Ji H L.Next-generation DNA sequencing[J].Nat Biotechnol,2008,26(10):1135.
[11]Glenn T C.Field guide to next-generation DNA sequencers[J].Mol Ecol Resour,2011,11(5):759.
[12]Song B H,Chen Z D,Wang X Q,et al.Sequence analysis of the ITS region of nuclear ribosomal DNA(nrDNA)in Chinese Amaranthus and its systematic utility[J].Acta Bot Sin,2000,42(11):1184.
[13]Yao H,Song J Y,Liu C,et al.Use of ITS2 region as the universal DNA barcode for plants and animals[J].PLoS ONE,2010,5(10):e13102.
[14]Fazekas A J,Burgess K S,Kesanakurti P R,et al.Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well[J].PLoS ONE,2008,3(7):e2802.
[15]Gao T,Sun Z Y,Yao H,et al.Identification of fabaceae plants using the DNA barcode matK[J].Planta Med,2011,77(1):92.
[16]Yao H,Song J Y,Ma X Y,et al.Identification of Dendrobium species by a candidate DNA barcode sequence:the chloroplast psb A-trnH intergenic region[J].Planta Med,2009,75(6):667.
[17]Li D Z,Gao L M,Li H T,et al.Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants[J].Proc Natl Acad Sci USA,2011,108(49):19641.
[18]Parks M,Cronn R,Liston A.Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes[J].BMC Biol,2009,7(1):84.
[19]Kress W J,Erickson D L.A two-locus global DNA barcode for land plants:the coding rbc L gene complements the non-coding trnH-psbA spacer region[J].PLoS ONE,2007,2(6):e508.
[20]Li F W,Kuo L Y,Rothfels C J,et al.RbcL and matK earn two thumbs up as the core DNA barcode for ferns[J].PLoS ONE,2011,6(10):e26597.
[21]Sogin M L,Morrison H G,Huber J A,et al.Microbial diversity in the deep sea and the under explored"rare biosphere"[J].Proc Natl Acad Sci USA,2006,103(32):12115.
[22]Lahaye R,Van der Bank M,Bogarin D,et al.DNA barcoding the floras of biodiversity hotspots[J].Proc Natl Acad Sci USA,2008,105(8):2923.
[23]Gillespie D E,Brady S F,Bettermann A D,et al.Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA[J].Appl Environ Microb,2002,68(9):4301.
[24]Venter J C,Remington K,Heidelberg J F,et al.Environmental genome shotgun sequencing of the Sargasso Sea[J].Science,2004,304(5667):66.
[25]Tyson G W,Chapman J,Hugenholtz P,et al.Community structure and metabolism through reconstruction of microbial genomes from the environment[J].Nature,2004,428(6978):37.
[26]Wagner M,Nielsen P H,Loy A,et al.Linking microbial community structure with function:fluorescence in situ hybridization-microautoradiography and isotope arrays[J].Curr Opin Biotech,2006,17(1):83.
[27]Gill S R,Pop M,De Boy R T,et al.Metagenomic analysis of the human distal gut microbiome[J].Science,2006,312(5778):1355.
[28]贺纪正,张丽梅,沈菊培,等.宏基因组学(metagenomics)的研究现状和发展趋势[J].环境科学学报,2008,28(2):209.
[29]Yu D W,Ji Y Q,Emerson B C,et al.Biodiversity soup:metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring[J].Methods Ecol Evol,2012,3(4):613.
[30]杨晨雪,季吟秋,王晓阳,等.基于18S r DNA的metabarcoading技术分析土壤小型动物多样性3种方法的比较[J].中国科学:生命科学,2012,42(12):993.
[31]Bienert F,De Danieli S,Miquel C,et al.Tracking earthworm communities from soil DNA[J].Mol Ecol,2012,21(8):2017.
[32]Thomsen P F,Kielgast J,Iversen L L,et al.Monitoring endangered freshwater biodiversity using environmental DNA[J].Mol Ecol,2012,21(11):2565.
[33]Silverio M S,Rodovalho V D,Bonetti A M,et al.Preliminary characterization of mitochondrial genome of Melipona scutellaris,a Brazilian stingless bee[J].Biomed Res Int,2014,2014(6):6.
[34]Yoccoz N G.The future of environmental DNA in ecology[J].Mol Ecol,2012,21(8):2031.
[35]Hiiesalu I,Opik M,Metsis M,et al.Plant species richness belowground:higher richness and new patterns revealed by nextgeneration sequencing[J].Mol Ecol,2012,21(8):2004.
[36]Wang L,Zhuang Y Y,Zhang H,et al.DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species[J].Harmful Algae,2014,31:100.
[37]Saunders G W.A DNA barcode examination of the red algal family Dumontiaceae in Canadian waters reveals substantial cryptic species diversity.1.The foliose Dilsea-Neodilsea complex and Weeksia[J].Botany-Botanique,2008,86(7):773.
[38]Passmore A J,Jarman S N,Swadling K M,et al.DNA as a dietary biomarker in antarctic krill,Euphausia superba[J].Mar Biotechnol,2006,8(6):686.
[39]Hulcr J,Mogia M,Isua B,et al.Host specificity of ambrosia and bark beetles(Col,Curculionidae:Scolytinae and Platypodinae)in a New Guinea rainforest[J].Ecol Entomol,2007,32(6):762.
[40]Bourlat S J,Nakano H,Akerman M,et al.Feeding ecology of Xenoturbella bocki(phylum Xenoturbellida)revealed by genetic barcoding[J].Mol Ecol Resour,2008,8(1):18.
[41]Valentini A,Miquel C,Nawaz M A,et al.New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing:the trnL approach[J].Mol Ecol Resour,2009,9(1):51.
[42]Garcia-Robledo C,Erickson D L,Staines C L,et al.Tropical plant-herbivore networks:reconstructing species interactions using DNA barcodes[J].PLoS ONE,2013,8(1):e52967.
[43]方强,黄双全.传粉网络的研究进展:网络的结构和动态[J].生物多样性,2012,20(3):300.
[44]Rafferty N E,Ives A R.Phylogenetic trait-based analyses of ecological networks[J].Ecology,2013,94(10):2321.
[45]田晓轩,刘杰,窦永杰,等.中成药大黄蛰虫丸的宏条形码基原鉴定[J].天津中医药,2014,31(4):234.
[46]Galimberti A,De Mattia F,Losa A,et al.DNA barcoding as a new tool for food traceability[J].Food Res Int,2013,50(1):55.
[47]Bruni I,Galimberti A,Caridi L,et al.A DNA barcoding approach to identify plant species in multiflower honey[J].Food Chem,2015,170:308.
[48]白虹,宁康,王长云.运用基于高通量测序和大数据挖掘的元基因组学方法分析中药制剂的物种成分[J].药学学报,2015,50(3):272.
[49]Mihalov J J,Marderosian A D,Pierce J C.DNA identification of commercial ginseng samples[J].J Agric Food Chem,2000,48(8):3744.
[50]刘光全,陶水华,李辉,等.虫草胶囊菌粉的鉴定与分析[J].中国食品卫生杂志,2009,21(5):418.
[51]王巍,邓赟,戴宇,等.全天麻胶囊中天麻的DNA鉴定[J].中药与临床,2015,6(1):38.
[52]崔占虎,蒋超,李旻辉,等.连翘败毒丸中原料药材的分子鉴别[J].药学学报,2013,48(4):590.
[53]Newmaster S G,Grguric M,Shanmughanandhan D,et al.DNAbarcoding detects contamination and substitution in North American herbal products[J].Bmc Med,2013,11:1.
[54]Ivanova N V,Kuzmina M L,Braukmann T W A,et al.Authentication of herbal supplements using next-generation sequencing[J].PLoS ONE,2016,11(5):e0168628.
[55]Coghlan M L,White N E,Murray D C,et al.Metabarcoding avian diets at airports:implications for birdstrike hazard management planning[J].Investigative Genetics,2012,4(1):27.
[56]Cheng X W,Su X Q,Chen X H,et al.Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing:the story for Liuwei Dihuang Wan[J].Sci Rep,2014,4:5147.
[57]Li Q,Sun Y,Guo H,et al.Quality control of the traditional Chinese medicine Ruyi jinhuang powder based on high-throughput sequencing and real-time PCR[J].Sci Rep,2018,8(1):8261.
[58]Coghlan M L,Maker G,Crighton E,et al.Combined DNA,toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine(TCM)[J].Sci Rep,2015,5:17475.
[59]Raclariu A C,Paltinean R,Vlase L,et al.Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding,TLC and HPLC-MS[J].Sci Rep,2017,7:1291.
[60]Raclariu A C,Mocan A,Popa M O,et al.Veronica officinalis product authentication using DNA metabarcoding and HPLC-MSreveals widespread adulteration with Veronica chamaedrys[J].Front Pharmacol,2017,8:378.
[61]宋经元,辛天怡,徐志超,等.一种基于smrt测序技术的中成药生物物种组成成分监测方法:中国106498050A[P].2017.
[62]Zheng X S,Zhang P,Liao B S,et al.A comprehensive quality evaluation system for complex herbal medicine using pacbio sequencing,PCR-denaturing gradient gel electrophoresis,and several chemical approaches[J].Front Plant Sci,2017,8:1578.
[63]张鹏.中药质量控制相关创新技术研究[D].北京:北京中医药大学,2017.
[64]赵莎,辛天怡,侯典云,等.党参药材及其混伪品的ITS/ITS2条形码鉴定研究[J].世界科学技术---中医药现代化,2013(3):421.
[65]Richardson R T,Lin C,Sponsler D B,et al.Application of ITS2metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem[J].Appl Plant Sci,2015,3(1):235.
[66]Zhou X,Li Y Y,Liu S L,et al.Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification[J].Gigascience,2013,2(1):4.
[2]EMA.Guideline on the assessment of clinical safety and efficacy in the preparation of European Union herbal monographs for wellestablished and traditional herbal medicinal products EMA/HMPC/104613/2005 Rev.1[EB/OL].(2016-07-12)[2018-04-24].http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/09/WC500212240.pdf.
[3]李浩,田侃,喻小勇,等.欧盟成员国植物药产品审评现状分析及启示[J].中草药,2016,47(24):4494.
[4]崔伟锋,王素花,王会丽,等.真实世界中医临床研究方法与实践[J].中国全科医学,2016,19(13):1586.
[5]钟丽丹.中成药上市后再评价的现状、挑战与展望[J].世界中医药,2017,12(6):1218.
[6]Hebert P D N,Cywinska A,Ball S L,et al.Biological identifications through DNA barcodes[J].P Roy Soc Lond B Bio,2003,270(1512):313.
[7]Li X W,Yang Y,Henry R J,et al.Plant DNA barcoding:from gene to genome[J].Biol Rev,2015,90(1):157.
[8]Chen S L,Pang X H,Song J Y,et al.A renaissance in herbal medicine identification:from morphology to DNA[J].Biotechnol Adv,2014,32(7):1237.
[9]陈士林,姚辉,韩建萍,等.中药材DNA条形码分子鉴定指导原则[J].中国中药杂志,2013,38(2):141.
[10]Shendure J,Ji H L.Next-generation DNA sequencing[J].Nat Biotechnol,2008,26(10):1135.
[11]Glenn T C.Field guide to next-generation DNA sequencers[J].Mol Ecol Resour,2011,11(5):759.
[12]Song B H,Chen Z D,Wang X Q,et al.Sequence analysis of the ITS region of nuclear ribosomal DNA(nrDNA)in Chinese Amaranthus and its systematic utility[J].Acta Bot Sin,2000,42(11):1184.
[13]Yao H,Song J Y,Liu C,et al.Use of ITS2 region as the universal DNA barcode for plants and animals[J].PLoS ONE,2010,5(10):e13102.
[14]Fazekas A J,Burgess K S,Kesanakurti P R,et al.Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well[J].PLoS ONE,2008,3(7):e2802.
[15]Gao T,Sun Z Y,Yao H,et al.Identification of fabaceae plants using the DNA barcode matK[J].Planta Med,2011,77(1):92.
[16]Yao H,Song J Y,Ma X Y,et al.Identification of Dendrobium species by a candidate DNA barcode sequence:the chloroplast psb A-trnH intergenic region[J].Planta Med,2009,75(6):667.
[17]Li D Z,Gao L M,Li H T,et al.Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants[J].Proc Natl Acad Sci USA,2011,108(49):19641.
[18]Parks M,Cronn R,Liston A.Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes[J].BMC Biol,2009,7(1):84.
[19]Kress W J,Erickson D L.A two-locus global DNA barcode for land plants:the coding rbc L gene complements the non-coding trnH-psbA spacer region[J].PLoS ONE,2007,2(6):e508.
[20]Li F W,Kuo L Y,Rothfels C J,et al.RbcL and matK earn two thumbs up as the core DNA barcode for ferns[J].PLoS ONE,2011,6(10):e26597.
[21]Sogin M L,Morrison H G,Huber J A,et al.Microbial diversity in the deep sea and the under explored"rare biosphere"[J].Proc Natl Acad Sci USA,2006,103(32):12115.
[22]Lahaye R,Van der Bank M,Bogarin D,et al.DNA barcoding the floras of biodiversity hotspots[J].Proc Natl Acad Sci USA,2008,105(8):2923.
[23]Gillespie D E,Brady S F,Bettermann A D,et al.Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA[J].Appl Environ Microb,2002,68(9):4301.
[24]Venter J C,Remington K,Heidelberg J F,et al.Environmental genome shotgun sequencing of the Sargasso Sea[J].Science,2004,304(5667):66.
[25]Tyson G W,Chapman J,Hugenholtz P,et al.Community structure and metabolism through reconstruction of microbial genomes from the environment[J].Nature,2004,428(6978):37.
[26]Wagner M,Nielsen P H,Loy A,et al.Linking microbial community structure with function:fluorescence in situ hybridization-microautoradiography and isotope arrays[J].Curr Opin Biotech,2006,17(1):83.
[27]Gill S R,Pop M,De Boy R T,et al.Metagenomic analysis of the human distal gut microbiome[J].Science,2006,312(5778):1355.
[28]贺纪正,张丽梅,沈菊培,等.宏基因组学(metagenomics)的研究现状和发展趋势[J].环境科学学报,2008,28(2):209.
[29]Yu D W,Ji Y Q,Emerson B C,et al.Biodiversity soup:metabarcoding of arthropods for rapid biodiversity assessment and biomonitoring[J].Methods Ecol Evol,2012,3(4):613.
[30]杨晨雪,季吟秋,王晓阳,等.基于18S r DNA的metabarcoading技术分析土壤小型动物多样性3种方法的比较[J].中国科学:生命科学,2012,42(12):993.
[31]Bienert F,De Danieli S,Miquel C,et al.Tracking earthworm communities from soil DNA[J].Mol Ecol,2012,21(8):2017.
[32]Thomsen P F,Kielgast J,Iversen L L,et al.Monitoring endangered freshwater biodiversity using environmental DNA[J].Mol Ecol,2012,21(11):2565.
[33]Silverio M S,Rodovalho V D,Bonetti A M,et al.Preliminary characterization of mitochondrial genome of Melipona scutellaris,a Brazilian stingless bee[J].Biomed Res Int,2014,2014(6):6.
[34]Yoccoz N G.The future of environmental DNA in ecology[J].Mol Ecol,2012,21(8):2031.
[35]Hiiesalu I,Opik M,Metsis M,et al.Plant species richness belowground:higher richness and new patterns revealed by nextgeneration sequencing[J].Mol Ecol,2012,21(8):2004.
[36]Wang L,Zhuang Y Y,Zhang H,et al.DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species[J].Harmful Algae,2014,31:100.
[37]Saunders G W.A DNA barcode examination of the red algal family Dumontiaceae in Canadian waters reveals substantial cryptic species diversity.1.The foliose Dilsea-Neodilsea complex and Weeksia[J].Botany-Botanique,2008,86(7):773.
[38]Passmore A J,Jarman S N,Swadling K M,et al.DNA as a dietary biomarker in antarctic krill,Euphausia superba[J].Mar Biotechnol,2006,8(6):686.
[39]Hulcr J,Mogia M,Isua B,et al.Host specificity of ambrosia and bark beetles(Col,Curculionidae:Scolytinae and Platypodinae)in a New Guinea rainforest[J].Ecol Entomol,2007,32(6):762.
[40]Bourlat S J,Nakano H,Akerman M,et al.Feeding ecology of Xenoturbella bocki(phylum Xenoturbellida)revealed by genetic barcoding[J].Mol Ecol Resour,2008,8(1):18.
[41]Valentini A,Miquel C,Nawaz M A,et al.New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing:the trnL approach[J].Mol Ecol Resour,2009,9(1):51.
[42]Garcia-Robledo C,Erickson D L,Staines C L,et al.Tropical plant-herbivore networks:reconstructing species interactions using DNA barcodes[J].PLoS ONE,2013,8(1):e52967.
[43]方强,黄双全.传粉网络的研究进展:网络的结构和动态[J].生物多样性,2012,20(3):300.
[44]Rafferty N E,Ives A R.Phylogenetic trait-based analyses of ecological networks[J].Ecology,2013,94(10):2321.
[45]田晓轩,刘杰,窦永杰,等.中成药大黄蛰虫丸的宏条形码基原鉴定[J].天津中医药,2014,31(4):234.
[46]Galimberti A,De Mattia F,Losa A,et al.DNA barcoding as a new tool for food traceability[J].Food Res Int,2013,50(1):55.
[47]Bruni I,Galimberti A,Caridi L,et al.A DNA barcoding approach to identify plant species in multiflower honey[J].Food Chem,2015,170:308.
[48]白虹,宁康,王长云.运用基于高通量测序和大数据挖掘的元基因组学方法分析中药制剂的物种成分[J].药学学报,2015,50(3):272.
[49]Mihalov J J,Marderosian A D,Pierce J C.DNA identification of commercial ginseng samples[J].J Agric Food Chem,2000,48(8):3744.
[50]刘光全,陶水华,李辉,等.虫草胶囊菌粉的鉴定与分析[J].中国食品卫生杂志,2009,21(5):418.
[51]王巍,邓赟,戴宇,等.全天麻胶囊中天麻的DNA鉴定[J].中药与临床,2015,6(1):38.
[52]崔占虎,蒋超,李旻辉,等.连翘败毒丸中原料药材的分子鉴别[J].药学学报,2013,48(4):590.
[53]Newmaster S G,Grguric M,Shanmughanandhan D,et al.DNAbarcoding detects contamination and substitution in North American herbal products[J].Bmc Med,2013,11:1.
[54]Ivanova N V,Kuzmina M L,Braukmann T W A,et al.Authentication of herbal supplements using next-generation sequencing[J].PLoS ONE,2016,11(5):e0168628.
[55]Coghlan M L,White N E,Murray D C,et al.Metabarcoding avian diets at airports:implications for birdstrike hazard management planning[J].Investigative Genetics,2012,4(1):27.
[56]Cheng X W,Su X Q,Chen X H,et al.Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing:the story for Liuwei Dihuang Wan[J].Sci Rep,2014,4:5147.
[57]Li Q,Sun Y,Guo H,et al.Quality control of the traditional Chinese medicine Ruyi jinhuang powder based on high-throughput sequencing and real-time PCR[J].Sci Rep,2018,8(1):8261.
[58]Coghlan M L,Maker G,Crighton E,et al.Combined DNA,toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine(TCM)[J].Sci Rep,2015,5:17475.
[59]Raclariu A C,Paltinean R,Vlase L,et al.Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding,TLC and HPLC-MS[J].Sci Rep,2017,7:1291.
[60]Raclariu A C,Mocan A,Popa M O,et al.Veronica officinalis product authentication using DNA metabarcoding and HPLC-MSreveals widespread adulteration with Veronica chamaedrys[J].Front Pharmacol,2017,8:378.
[61]宋经元,辛天怡,徐志超,等.一种基于smrt测序技术的中成药生物物种组成成分监测方法:中国106498050A[P].2017.
[62]Zheng X S,Zhang P,Liao B S,et al.A comprehensive quality evaluation system for complex herbal medicine using pacbio sequencing,PCR-denaturing gradient gel electrophoresis,and several chemical approaches[J].Front Plant Sci,2017,8:1578.
[63]张鹏.中药质量控制相关创新技术研究[D].北京:北京中医药大学,2017.
[64]赵莎,辛天怡,侯典云,等.党参药材及其混伪品的ITS/ITS2条形码鉴定研究[J].世界科学技术---中医药现代化,2013(3):421.
[65]Richardson R T,Lin C,Sponsler D B,et al.Application of ITS2metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem[J].Appl Plant Sci,2015,3(1):235.
[66]Zhou X,Li Y Y,Liu S L,et al.Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification[J].Gigascience,2013,2(1):4.