基于位点特异性PCR鉴别多花黄精及其混淆品
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摘要
为了建立快速区分多花黄精及其混淆品的鉴别方法,本研究采用位点特异性PCR方法,通过对多花黄精及其混淆品的psb A-trnH片段进行扩增。同源比对后根据其变异位点设计特异性鉴别引物,分析多花黄精及其混淆品的位点特异性差异情况。结果表明:本研究设计的特异性鉴定引物,在一个PCR反应中多花黄精能扩增出329 bp的条带,而混淆品不能扩增出条带,从而实现了正伪品的鉴别。该方法有效区别多花黄精与其混淆品品种,经多次试验,重复性较好。本研究为快速区分多花黄精及其混淆品提供了有效办法。
In order to establish a rapid identification method for Polygonatum cyrtonema and its adulterants,site-specific PCR was used to amplify the psbA-trnH fragment of Polygonatum cyrtonema and its adulterants. After homology comparison, specific primers were designed according to their mutation sites, and the site specificity differences between Polygonatum cyrtonema and its adulterants were analyzed. The results showed that the specific identification primers designed in this study could amplify 329 bp bands of Polygonatum cyrtonema in a single PCR reaction, while the adulterants could not amplify the bands, thus realizing the identification of authentic and counterfeit products. This method could effectively distinguish Polygonatum cyrtonema and its adulterants. After many experiments, the method had good repeatability. This study could provide an effective method for quickly distinguishing Polygonatum cyrtonema and its adulterants.
In order to establish a rapid identification method for Polygonatum cyrtonema and its adulterants,site-specific PCR was used to amplify the psbA-trnH fragment of Polygonatum cyrtonema and its adulterants. After homology comparison, specific primers were designed according to their mutation sites, and the site specificity differences between Polygonatum cyrtonema and its adulterants were analyzed. The results showed that the specific identification primers designed in this study could amplify 329 bp bands of Polygonatum cyrtonema in a single PCR reaction, while the adulterants could not amplify the bands, thus realizing the identification of authentic and counterfeit products. This method could effectively distinguish Polygonatum cyrtonema and its adulterants. After many experiments, the method had good repeatability. This study could provide an effective method for quickly distinguishing Polygonatum cyrtonema and its adulterants.
引文
Chen S.L.,2015,Standard DNA barcodes of Chinese material medical in Chinese pharmacopoeia,Science Press,Beijing,China,pp.53-493(陈士林,2015,中国药典中药材DNA条形码标准序列,科学出版社,中国,北京,pp.53-493)
Chen S.L.,Yao H.,Han J.P.,Xin T.Y.,Pang X.H.,Shi L.C.,Luo K.,Song J.Y.,Hou D.Y.,Shi S.M.,and Qian Z.Z.,2013,Principles for molecular identification of traditional Chinese material medical using DNA barcoding,Zhongguo Zhongyao Zazhi(China Journal of Chinese Material Medical),38(2):141-148(陈士林,姚辉,韩建萍,辛天怡,庞晓慧,石林春,罗焜,宋经元,侯典云,石上梅,钱忠直,2013,中药材DNA条形码分子鉴定指导原则,中国中药杂志,38(2):141-148)
Ding X.Y.,Wang Z.T.,Zhou K.Y.,Xu L.S.,Xu H.,and Wang Y.Q.,2003,Allele-specific primers for diagnostic PCR authentication of Dendrobium officinale,Planta Med.,69(6):587-588
Gao B.M.,Pan K.,Tian J.P.,Wei N.,and Zhang J.Q.,2015,Establishment and optimization of ITS-PCR system in SouthChina medicine Alpinia oxyphylla miquel,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):1061-1066(高炳淼,潘坤,田建平,魏娜,张俊清,2015,南药益智ITS-PCR体系的建立与优化,基因组学与应用生物学,34(5):1061-1066)
Gao H.,Zhang Z.B.,Wang W.L.,Zhang J.,Lv L.Y.,and Huang Y.,2008,Bidirectional PCR amplification of specific alleles:a novel method of SNP typing for rapidly and simultaneously differentiating homozygotes and heterozygotes,Yixue Fenzi Shengwuxue Zazhi(Journal of Medical Molecular Biology),5(6):524-528(高红,张志波,王维林,张娟,吕良英,黄英,2008,双向等位基因特异性PCR快速区分纯合子和杂合子SNP分型的新方法,医学分子生物学杂志,5(6):524-528)
Han J.P.,Song J.Y.,Yao H.,Chen X.C.,and Chen S.L.,2012,Comparison of DNA barcoders in identifying medicinal ma terials,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),37(8):1056-1061(韩建萍,宋经元,姚辉,陈晓辰,陈士林,2012,中药材DNA条形码鉴定的基因序列比较,中国中药杂志,37(8):1056-1061)
Huang L.Q.,and Yuan Y.,eds.,2014,Molecular identification of traditional Chinese medicine(TCM)a laboratory manual,Shanghai Scientific and Technical Publishers,Shanghai,China,pp.25-26(黄璐琦,袁媛,编著,2014,中药分子鉴定操作指南,上海科学技术出版社,中国,上海,pp.25-26)
Jigden B.,Wang H.,Kyum K.M.,Kim Y.J.,Gyo I.J.,and Yang D.C.,2010,Authentication of the oriental medicinal plant Ligusticum tenuissimum(Nakai)Kitagawa(Korean Go-Bon)by multiplex PCR,Planta Med.,76(6):648-651
Li X.X.,Ding X.Y.,Chu B.H.,Ding G.,Gu S.,Qiang L.,Wang Y.,and Zhou Q.,2007,Molecular authentication of Alisma orientale by PCR-RFLP and ARMS,Planta Med.,73(1):67-70
Liu Z.Q.,Wang Y.Q.,Zhou K.Y.,Han D.M.,Yang X.G.,and Liu X.H.,2001,Authentication of Chinese crude drug,Gecko,by allele-specific diagnostic PCR,Planta Med.,67(4):385-387
Lu S.,Ding X.Y.,Han L.,Han D.M.,Yang X.G.,and Liu X.H.,2010,Confirming the genetic identity of Dendrobium fimbriatum using an amplification refractory mutation system(ARMS),Plant Mol.Biol.Rep.,28(4):712-716
Qian L.,Ging G.,Zhou Q.,Feng Z.Y.,Ding X.Y.,Gu S.,Wang Y.,Li X.X.,and Chu B.H.,2008,Molecular authentication of Dendrobium loddigesii Rolfe by amplification refractory mutation system(ARMS),Planta Med.,74(4):470-473
Wang H.,Kim M.K.,Kim Y.J.,Lee H.N.,Jin H.Z.,Chen J.Y.,and Yang D.C.,2012,Molecular authentication of the oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers,Gene,494(1):92-95
Wang H.,Sun H.,Kwon W.S.,Jin H.Z.,and Yang D.C.,2010,APCR-based SNP marker for specific authentication of Korean ginseng(panax ginseng)cultivar"Chunpoong",Mol.Biol.Rep.,37(2):1053-1057
Xiao P.G.,ed.,2002,Modern Chinese material medica,Chemical Industry Press,Beijing,China,pp.902-910(肖培根主编,2002,新编中药志,化学工业出版社,中国,北京,pp.902-910)
Zhang J.Z.,2013,The study on DNA Bar coding of Polygonatum sibiricum Red.and Polygonatum cyrtonema Hua,Thesis for M.S.,Hunan University of Chinese Medicine,Supervisor:Li S.X.,pp.20-27(张家曾,2013,黄精与多花黄精的DNA条形码研究,硕士学位论文,湖南中医药大学,导师:李顺祥,pp.20-27)
Zhou Y.,2007,Identification of Rhizoma polygonati and its scavenging effect on superoxide anion free-radical,Tianjin Medical University,Thesis for M.S.,Supervisor:Yao Z.,pp.31-39(周晔,2007,中药黄精真伪鉴定及抗超氧阴离子自由基的研究,硕士学位论文,天津医科大学,导师:姚智,pp.31-39)
Chen S.L.,Yao H.,Han J.P.,Xin T.Y.,Pang X.H.,Shi L.C.,Luo K.,Song J.Y.,Hou D.Y.,Shi S.M.,and Qian Z.Z.,2013,Principles for molecular identification of traditional Chinese material medical using DNA barcoding,Zhongguo Zhongyao Zazhi(China Journal of Chinese Material Medical),38(2):141-148(陈士林,姚辉,韩建萍,辛天怡,庞晓慧,石林春,罗焜,宋经元,侯典云,石上梅,钱忠直,2013,中药材DNA条形码分子鉴定指导原则,中国中药杂志,38(2):141-148)
Ding X.Y.,Wang Z.T.,Zhou K.Y.,Xu L.S.,Xu H.,and Wang Y.Q.,2003,Allele-specific primers for diagnostic PCR authentication of Dendrobium officinale,Planta Med.,69(6):587-588
Gao B.M.,Pan K.,Tian J.P.,Wei N.,and Zhang J.Q.,2015,Establishment and optimization of ITS-PCR system in SouthChina medicine Alpinia oxyphylla miquel,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):1061-1066(高炳淼,潘坤,田建平,魏娜,张俊清,2015,南药益智ITS-PCR体系的建立与优化,基因组学与应用生物学,34(5):1061-1066)
Gao H.,Zhang Z.B.,Wang W.L.,Zhang J.,Lv L.Y.,and Huang Y.,2008,Bidirectional PCR amplification of specific alleles:a novel method of SNP typing for rapidly and simultaneously differentiating homozygotes and heterozygotes,Yixue Fenzi Shengwuxue Zazhi(Journal of Medical Molecular Biology),5(6):524-528(高红,张志波,王维林,张娟,吕良英,黄英,2008,双向等位基因特异性PCR快速区分纯合子和杂合子SNP分型的新方法,医学分子生物学杂志,5(6):524-528)
Han J.P.,Song J.Y.,Yao H.,Chen X.C.,and Chen S.L.,2012,Comparison of DNA barcoders in identifying medicinal ma terials,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),37(8):1056-1061(韩建萍,宋经元,姚辉,陈晓辰,陈士林,2012,中药材DNA条形码鉴定的基因序列比较,中国中药杂志,37(8):1056-1061)
Huang L.Q.,and Yuan Y.,eds.,2014,Molecular identification of traditional Chinese medicine(TCM)a laboratory manual,Shanghai Scientific and Technical Publishers,Shanghai,China,pp.25-26(黄璐琦,袁媛,编著,2014,中药分子鉴定操作指南,上海科学技术出版社,中国,上海,pp.25-26)
Jigden B.,Wang H.,Kyum K.M.,Kim Y.J.,Gyo I.J.,and Yang D.C.,2010,Authentication of the oriental medicinal plant Ligusticum tenuissimum(Nakai)Kitagawa(Korean Go-Bon)by multiplex PCR,Planta Med.,76(6):648-651
Li X.X.,Ding X.Y.,Chu B.H.,Ding G.,Gu S.,Qiang L.,Wang Y.,and Zhou Q.,2007,Molecular authentication of Alisma orientale by PCR-RFLP and ARMS,Planta Med.,73(1):67-70
Liu Z.Q.,Wang Y.Q.,Zhou K.Y.,Han D.M.,Yang X.G.,and Liu X.H.,2001,Authentication of Chinese crude drug,Gecko,by allele-specific diagnostic PCR,Planta Med.,67(4):385-387
Lu S.,Ding X.Y.,Han L.,Han D.M.,Yang X.G.,and Liu X.H.,2010,Confirming the genetic identity of Dendrobium fimbriatum using an amplification refractory mutation system(ARMS),Plant Mol.Biol.Rep.,28(4):712-716
Qian L.,Ging G.,Zhou Q.,Feng Z.Y.,Ding X.Y.,Gu S.,Wang Y.,Li X.X.,and Chu B.H.,2008,Molecular authentication of Dendrobium loddigesii Rolfe by amplification refractory mutation system(ARMS),Planta Med.,74(4):470-473
Wang H.,Kim M.K.,Kim Y.J.,Lee H.N.,Jin H.Z.,Chen J.Y.,and Yang D.C.,2012,Molecular authentication of the oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers,Gene,494(1):92-95
Wang H.,Sun H.,Kwon W.S.,Jin H.Z.,and Yang D.C.,2010,APCR-based SNP marker for specific authentication of Korean ginseng(panax ginseng)cultivar"Chunpoong",Mol.Biol.Rep.,37(2):1053-1057
Xiao P.G.,ed.,2002,Modern Chinese material medica,Chemical Industry Press,Beijing,China,pp.902-910(肖培根主编,2002,新编中药志,化学工业出版社,中国,北京,pp.902-910)
Zhang J.Z.,2013,The study on DNA Bar coding of Polygonatum sibiricum Red.and Polygonatum cyrtonema Hua,Thesis for M.S.,Hunan University of Chinese Medicine,Supervisor:Li S.X.,pp.20-27(张家曾,2013,黄精与多花黄精的DNA条形码研究,硕士学位论文,湖南中医药大学,导师:李顺祥,pp.20-27)
Zhou Y.,2007,Identification of Rhizoma polygonati and its scavenging effect on superoxide anion free-radical,Tianjin Medical University,Thesis for M.S.,Supervisor:Yao Z.,pp.31-39(周晔,2007,中药黄精真伪鉴定及抗超氧阴离子自由基的研究,硕士学位论文,天津医科大学,导师:姚智,pp.31-39)