快速PCR方法在金银花真伪鉴别中的应用Rapid PCR authentication Lonicera japanica
蒋超;侯静怡;黄璐琦;袁媛;陈敏;金艳;
JIANG Chao;HOU Jing-yi;HUANG Lu-qi;YUAN Yuan;CHEN Min;JIN Yan;Institute of Resource Ecology and Traditional Chinese Medicine Resources,Beijing Normal University;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;
摘要(Abstract):
建立简单快速的金银花真伪分子鉴别方法。该研究依据金银花trn L-trn F 625位G/T SNP位点设计快速位点特异性PCR引物,优化位点特异性PCR条件,对金银花及其9种混淆品进行扩增及检测。当在87℃预变性1 min;87℃变性5s,68℃延伸5 s,30个循环时,通过加入SYBR Green I染料染色,金银花样品显绿色荧光,混淆品无荧光。整个PCR反应可在30 min内完成。该研究结果说明快速位点特异性PCR能简单快速鉴别金银花及其混淆品。
To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trn Ltrn F 625 G / T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 × SYBR Green I was added in the PCR product of87 ℃ initial denatured 1 min; 87 ℃ denatured 5 s,68 ℃ annealing 5 s,30 cycle; L. japanica visualize strong green fluorescence under365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.
关键词(KeyWords):
快速PCR;金银花;分子鉴定;荧光检测
rapid PCR;Lonicera japonica;molecular authentication;fluorescence
基金项目(Foundation): 国家自然科学基金项目(81373959);; 中医药行业科研专项(201407003)
作者(Author):
蒋超;侯静怡;黄璐琦;袁媛;陈敏;金艳;
JIANG Chao;HOU Jing-yi;HUANG Lu-qi;YUAN Yuan;CHEN Min;JIN Yan;Institute of Resource Ecology and Traditional Chinese Medicine Resources,Beijing Normal University;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;
Email:
DOI:
参考文献(References):
- [1]Yap E P,Mcgee J O.Short PCR product yields improved by lower denaturation temperatures[J].Nucleic Acids Res,1991,19(7):1713.
- [2]Wittwer C T,Fillmore G C,Garling D J.Minimizing the time required for DNA amplification by efficient heat transfer to small samples[J].Anal Biochem,1990,186(2):328.
- [3]Wittwer C T,Garling D J.Rapid cycle DNA amplification:time and temperature optimization[J].Biotechniques,1991,10(1):76.
- [4]Aboud M,Oh H H,Mccord B.Rapid direct PCR for forensic genotyping in under 25 min[J].Electrophoresis,2013,34(11):1539.
- [5]Yip P Y,Chau C F,Mak C Y,et al.DNA methods for identification of Chinese medicinal materials[J].Chin Med,2007,2(9):9.
- [6]Heubl G U N.New aspects of DNA-based authentication of Chinese medicinal plants by molecular biological techniques[J].Planta Med,2010,76(17):1963.
- [7]蒋超,张雅华,陈敏,等.基于双向位点特异性PCR的金银花真伪鉴别方法研究[J].中国中药杂志,2012(24):3752.
- [8]蒋超,黄璐琦,袁媛,等.使用碱裂解法快速提取药材DNA方法的研究[J].药物分析杂志,2013(7):1081.
- [9]钟罗宝,陈谷.一种适用于大规模转基因作物PCR检测的简易DNA提取方法[J].现代食品科技,2008(8):794.
- [10]高红,张志波,王维林,等.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[J].医学分子生物学杂志,2008(6):524.
- [11]Lee H Y,Park M J,Kim N Y,et al.Rapid direct PCR for ABO blood typing[J].J Forensic Sci,2011,56(s1):S179.
- [12]French D J,Archard C L,Andersen M T,et al.Ultra-rapid DNA analysis using Hy BeaconTMprobes and direct PCR amplification from saliva[J].Mol Cell Probe,2002,16(5):319.
- [13]Wernike K,Beer M,Hoffmann B.Rapid detection of foot-andmouth disease virus,influenza A virus and classical swine fever virus by high-speed real-time RT-PCR[J].J Virol Methods,2013,193(1):50.
- [14]Wheeler E K,Hara C A,Frank J,et al.Under-three minute PCR:probing the limits of fast amplification[J].Analyst,2011,136(18):3707.
- [15]Bergamo E,Chiapolino G,Lignitto L,et al.Evaluation of fast PCR reagents for rapid and sensitive detection of human herpesvirus 8[J].J Virol Methods,2012,181(1):125.
- [16]Zhang L,Dang F,Kaji N,et al.Fast extraction,amplification and analysis of genes from human blood[J].J Chromatogr A,2006,1106(1/2):175.
- [17]袁媛,蒋超,黄璐琦.中药材分子鉴别现场运用的策略与实践[J].中国中药杂志,2013,38(16):2553.
- [18]Wittwer C T,Marshall B C,Reed G H,et al.Rapid cycle allele-specific amplification:studies with the cystic fibrosis delta F508 locus[J].Clin Chem,1993,39(5):804.
- [19]张文超.聚合酶链反应(PCR)技术与基因扩增分析仪器(PCR仪)[J].生命科学仪器,2005(3):13.
- [20]杨文超,张晓东.快速PCR研究进展[J].中国生物工程杂志,2007(4):99.
- [21]Hashimoto M,Chen P C,Mitchell M W,et al.Rapid PCR in a continuous flow device[J].Lab Chip,2004,4(6):638.
- [22]Neuzil P,Novak L,Pipper J,et al.Rapid detection of viral RNA by a pocket-size real-time PCR system[J].Lab Chip,2010,10(19):2632.
- [23]Agrawal N,Hassan Y A,Ugaz V M.A pocket-sized convective PCR thermocycler[J].Angew Chem Int Ed Engl,2007,46(23):4316.
- 蒋超
- 侯静怡
- 黄璐琦
- 袁媛
- 陈敏
- 金艳
JIANG Chao- HOU Jing-yi
- HUANG Lu-qi
- YUAN Yuan
- CHEN Min
- JIN Yan
- Institute of Resource Ecology and Traditional Chinese Medicine Resources
- Beijing Normal University
- State Key Laboratory of Dao-di Herbs
- National Resource Center for Chinese Materia Medica
- China Academy of Chinese Medical Sciences
- 蒋超
- 侯静怡
- 黄璐琦
- 袁媛
- 陈敏
- 金艳
JIANG Chao- HOU Jing-yi
- HUANG Lu-qi
- YUAN Yuan
- CHEN Min
- JIN Yan
- Institute of Resource Ecology and Traditional Chinese Medicine Resources
- Beijing Normal University
- State Key Laboratory of Dao-di Herbs
- National Resource Center for Chinese Materia Medica
- China Academy of Chinese Medical Sciences