人参属中药材高分辨率熔解曲线鉴定及其方法学考察

目的：建立基于高分辨率熔解曲线鉴别人参属中药材的方法，并进行系统性方法学考察。方法：采集不同产地的人参属中药材人参、西洋参、三七以及市场上常见混伪品竹节参、珠子参、羽叶三七。所有样品提取总DNA，筛选合适的引物，构建人参属正品中药材熔解曲线。同时对不同比例混合样品进行了鉴别，对该方法的灵敏性与特异性、重复性、稳健性、检出限进行了系统性考察。结果：选择psbA-F/trnH-R引物，在模板质量浓度1.6~200 ng·μL^-1，退火温度为54~60℃，引物浓度为0.1~0.3 μmol·L^-1范围内，对人参属中药材共75份样品进行高分辨熔解曲线分析，人参、西洋参、三七、羽叶三七、竹节参、珠子参等均获得正确稳定的分析结果。结论：高分辨熔解曲线依据分型分析可以区分人参属中药材，并对其混伪品进行检测。在此基础上，本文提出了中药分子鉴定方法学研究标准程序，为进一步规范中药分子鉴定方法学研究提供依据。

Objective: To establish a method for the identification of Panax species based on high resolution melting(HRM),and to carry out systematic methodological study.Methods: Panax species such as:Panax ginseng,Panax quinquefolium,Panax notoginseng,Panax japonicus,Panax japonicas var. major,Panax pseudoginseng var. bipinnatifidus were collected.The DNA of all the collected samples were extracted;the suitable primers were selected and the melting curve for Panax species was developed.The fakes and adulterants of different mix were also tested.Molecular identification,including the sensitivity,specificity,repeatability,robustness,and the limit of detection was systematically investigated.Results: A total of 75 samples of Panax ginseng were selected by using primer psbA-F/trnH-R.The template concentration was 1.6-200 ng·μL^-1,primer concentration for the psbA-F/trnH-R primer was 0.1-0.3 μmol·L^-1,and annealing temperature was 54-60℃.The analysis of high-resolution melting curve on Panax species can obtain the correct stable identification.Conclusion: High resolution melting curve analysis can distinguish Panax species by genotyping,and its adulterants can be detected.On this basis,this paper presents a standard method for the molecular identification of traditional Chinese medicine,which provides a basis for further standardizing the molecular identification of traditional Chinese medicine.

[1] ZUO Y,CHEN Z,KONDO K,et al.DNA barcoding of Panax species[J].Planta Med,2011,77(2):182
[2] ZHU S,FUSHIMI H,CAI S,et al.Species identification from ginseng drugs by multiplex amplification refractory mutation system(MARMS)[J].Planta Med,2004,70(2):189
[3] 王川易,郭宝林,肖培根.中药分子鉴定方法评述[J].中国中药杂志,2011,36(3):237 WANG CY,GUO BL,XIAO PG.Molecuar methods for authentication of Chinese medicinal materials[J].China J Chin Mater Med,2011,36(3):237
[4] HEUBL G.New aspects of DNA-based authentication of Chinese medicinal plants by molecular biological techniques[J].Planta Med,2010,76(17):1963
[5] SHAW PC,BUT PP. Authentication of Panax species and their adulterants by random-primed polymerase chain reaction[J].Planta Med,1995,61(5):466
[6] HA WY,SHAW PC,LIU J,et al.Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism(AFLP) and directed amplification of minisatellite region DNA(DAMD)[J].J Agric Food Chem,2002,50(7):1871
[7] DIAO Y,LIN XM,LIAO CL,et al.Authentication of Panax ginseng from its adulterants by PCR-RFLP and ARMS[J].Planta Med,2009,75(5):557
[8] UM JY,CHUNG HS,KIM MS,et al.Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP[J].Biol Pharm Bull,2001,24(8):872
[9] WANG J,HA WY,NGAN FN,et al.Application of sequence characterized amplified region(SCAR) analysis to authenticate Panax species and their adulterants[J].Planta Med,2001,67(8):781
[10] WANG H,KIM MK,KWON WS,et al.Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region[J].J Pharm Biomed Anal,2011,55(5):972
[11] 崔光红,黄璐琦,唐晓晶,等.获取人参、西洋参特定序列位点(STS)标记的新方法[J].中国中药杂志,2007,32(11):1012 CUI GH,HUANG LQ,TANG XJ,et al.Application of multiplex allele-specific PCR for authentication of Panax ginseng and P. quinquefolius[J].China J Chin Mater Med,2007,32(11):1012
[12] BANG KH,JO IH,KIM YC,et al.Molecular identification of Korean ginseng cultivars(Panax ginseng CA Mey.) using peptide nucleic acid(PNA) microarray[J].Korean J Med Crop Sci,2012,20(5):387
[13] LEE JW,BANG KH,CHOI JJ,et al.Development of peptide nucleic acid(PNA) microarray for identification of Panax species based on the nuclear ribosomal internal transcribed spacer(ITS) and 5.8 S rDNA regions[J].Genes Genom,2010,32(5):463
[14] 任保青,陈之端.植物DNA条形码技术[J].植物学报,2010,45(1):1 REN BQ,CHEN ZD.DNA barcoding plant life[J].Chin Bull Bot,2010,45(1):1
[15] KRESS WJ,WURDACK KJ,ZIMMER EA,et al.Use of DNA barcodes to identify flowering plants[J].Proc Natl Acad Sci USA,2005,102(23):8369
[16] LI D,GAO L,LI H,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].P Natl Acad Sci USA,2011,108(49):19641
[17] CHEN S,YAO H,HAN J,et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PloS One,2010,5(1):e8613
[18] HOLLINGSWORTH PM,FORREST LL,SPOUGE JL,et al.A DNA barcode for land plants[J].P Natl Acad Sci USA,2009,106(31):12794
[19] LI M,CAO H,BUT PP,et al.Identification of herbal medicinal materials using DNA barcodes[J].J Syst Evol,2011,49(3):271
[20] LUO K,CHEN S,CHEN K,et al.Assessment of candidate plant DNA barcodes using the Rutaceae family[J].Sci China Life Sci,2010,53(6):701
[21] TECHEN N,PARVEEN I,PAN Z,et al.DNA barcoding of medicinal plant material for identification[J].Curr Opin Biotech,2014,25(2):103
[22] CHEN X,LIAO B,SONG J,et al.A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding[J].Gene,2013,530(1):39
[23] RIRIE KM,RASMUSSEN RP,WITTWER CT.Product differentiation by analysis of DNA melting curves during the polymerase chain reaction[J].Anal Biochem,1997,245(2):154
[24] DRUML B,CICHN MM.High resolution melting(HRM) analysis of DNA-its role and potential in food analysis[J].Food Chem,2014,158(9):245
[25] ARTHOFER W,STEINER FM,SCHLICK SBC. Rapid and cost-effective screening of newly identified microsatellite loci by high-resolution melting analysis[J].Mol Genet Genom,2011,286(3-4):225
[26] WOJDACZ TK,DOBROVIC A.Methylation-sensitive high resolution melting(MS-HRM):a new approach for sensitive and high-throughput assessment of methylation[J].Nucl Acids Res,2007,35(12):e41
[27] MARTINO A,MANCUSO T,ROSSI AM.Application of high-resolution melting to large-scale,high-throughput SNP genotyping:a comparison with the TaqMan method[J].J Biomol Screen,2010,15(6):623
[28] JAAKOLA L,SUOKAS M,HAGGMAN H.Novel approaches based on DNA barcoding and high-resolution melting of amplicons for authenticity analyses of berry species[J].Food Chem,2010,123(2):494
[29] GANOPOULOS I,MADESIS P,DARZENTAS N,et al.Barcode high pesolution melting(Bar-HRM) analysis for detection and quantification of PDO "Fava Santorinis"(Lathyrus clymenum) adulterants[J].Food Chem,2012,133(2):505
[30] FARIA MA,MAGALHAES A,NUNES ME,et al.High resolution melting of trnL amplicons in fruit juices authentication[J].Food Control,2013,33(1):136
[31] MADESIS P,GANOPOULOS I,ANAGNOSTIS A,et al.The application of Bar-HRM(Barcode DNA-high resolution melting) analysis for authenticity testing and quantitative detection of bean crops(Leguminosae) without prior DNA purification[J].Food Control,2012,25(2):576
[32] GANOPOULOS I,BAZAKOS C,MADESIS P,et al.Barcode DNA high-resolution melting(Bar-HRM) analysis as a novel close-tubed and accurate tool for olive oil forensic use[J].J Sci Food Agric,2013,93(9):2281
[33] 崔光红,唐晓晶,黄璐琦.含淀粉及多糖类中药材DNA的提取方法研究[J].中国中药杂志,2006,31(16):1365 CUI GH,TANG XJ,HUANG LQ.The studies for DNA extraction from Chinese herbal medicines which contain polysaccharides and starch[J].China J Chin Mater Med,2006,31(16):1365
[34] NORAMBUENA PA,COPELAND JA,KRENKOVA P,et al.Diagnostic method validation:high resolution melting(HRM) of small amplicons genotyping for the most common variants in the MTHFR gene[J].Clin Biochem,2009,42(12):1308
[35] 陈康,蒋超,袁媛,等.高分辨率熔解曲线技术及其在中药DNA分子鉴定中的应用[J].药学学报,2015,50(12):1581 CHEN K,JIANG C,YUAN Y,et al.High resolution melting and its application in identity study of traditional Chinese medicine[J].Acta Pharm Sin,2015,50(12):1581
[36] 鲁静,付凌燕,王旭.质量分析方法验证中检出限和定量限测定方法探讨[J].中国药品标准,2012,13(1):33 LU J,FU LY,WANG X.Discussion on the detecting methods for limit of detection and limit of quantification in validation of analytical method[J].Drug Stand China,2012,13(1):33
[37] PRENCE EM.A practical guide for the validation of genetic tests[J].Genet Test,1999,3(2):201
[38] EP6-A,Evaluation of the Linearity of Quantitative Measurement Procedures:A Statistical Approach;Approved Guideline[S].2003
[39] EP15-A,User Demonstration of Performance for Precision and Accuracy;Approved Guideline[S].2001