基于双向位点特异性PCR的金银花真伪鉴别方法研究Authentication of Lonicera japonica using bidirectional PCR amplification of specific alleles
蒋超;张雅华;陈敏;袁媛;林淑芳;吴志刚;
JIANG Chao,ZHANG Ya-hua,CHEN Min,YUAN Yuan*,LIN Shu-fang,WU Zhi-gang(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
摘要(Abstract):
目的:筛选获得金银花真伪鉴别SNP位点并建立双向位点特异性PCR方法,用于鉴别金银花和其常见伪品以及两者的混杂品。方法:通过对GenBank收录的忍冬属植物叶绿体trnL-trnF序列进行对比分析,获得金银花真伪鉴别SNP位点;并依据该SNP位点设计特异性引物,对84份金银花基原植物及其市售饮片、伪品进行双向位点特异性PCR扩增,并根据真伪品的特异性条带进行金银花药材鉴别。结果:退火温度为61℃时,正品均出现468 bp的条带,红腺忍冬、华南忍冬、灰毡毛忍冬、黄褐毛忍冬、水忍冬、金银忍冬、郁香忍冬、新疆忍冬、繁果忍冬等9个伪品均出现324 bp的条带,在正品DNA中掺入5%以上伪品时同时出现正品和伪品条带。结论:双向位点特异性PCR可以鉴别金银花真伪品及两者的混杂样品。
Objective: To identify SNP in flos Lonicerae,and authenticate Lonicera japonica from its adulterants and the mixture by using bidirectional PCR amplification of specific alleles(Bi-PASA).Method: SNP of L.japonica and its adulterants was identified by using ClustulW to align trnL-trnF sequences of the Lonicera genus from GenBank database.Bi-PASA primer was designed and the PCR reaction systems including annealing temperature optimized.Optimized result was performed in 84 samples to authenticate L.japonica,its adulterants and the mixture.Result: When the annealing temperature was 61 ℃,DNA from L.japonica would be amplified 468 bp whereas PCR products from all of the 9 adulterants were 324 bp.The established method also can detect 5% of intentional adulteration DNA into L.japonica.Conclusion: The Bi-SPASA could authenticate L.japonica from its adulterants and the mixture.
关键词(KeyWords):
双向位点特异性PCR;金银花;分子鉴定
bidirectional PCR amplification of specific alleles;Lonicera japonica;molecular authentication
基金项目(Foundation): 国家自然科学基金项目(81001605);; 中药制药过程新技术国家重点实验室开放基金项目(SKL2010M0101)
作者(Author):
蒋超;张雅华;陈敏;袁媛;林淑芳;吴志刚;
JIANG Chao,ZHANG Ya-hua,CHEN Min,YUAN Yuan*,LIN Shu-fang,WU Zhi-gang(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
Email:
DOI:
参考文献(References):
- [1]Zheng Q,Yue P,Wu B,et al.Pharmacokinetics comparativestudy of a novel Chinese traditional herbal formula and its com-patibility[J].J Ethnopharmacol,2011,137(1):221.
- [2]Ryu K H,Rhee H I,Kim J H,et al.Anti-inflammatory and an-algesic activities of SKLJI,a highly purified and injectable herbalextract of Lonicera japonica[J].Biosci Biotechnol Biochem,2010,74(10):2022.
- [3]Rahman A,Kang S C.In vitro control of food-borne and foodspoilage bacteria by essential oil and ethanol extracts of Lonicerajaponica Thunb[J].Food Chem,2009,116(3):670.
- [4]Wu L.Effect of chlorogenic acid on antioxidant activity of FlosLonicerae extracts[J].J Zhejiang Univ Sci B,2007(9):573.
- [5]Lee J H,Ko W S,Kim Y H,et al.Anti-inflammatory effect ofthe aqueous extract from Lonicera japonica flower is related to in-hibition of NF-kappaB activation through reducing I-kappa Bal-pha degradation in rat liver[J].Int J Mol Med,2001,7(1):79.
- [6]中国药典.一部[S].2005:205.
- [7]董桂灵,胡尚钦,陈晓敏,等.不同金银花种质资源ISSR遗传多样性研究[J].四川大学学报:自然科学版,2009,46(6):1833.
- [8]Peng X,Li W,Wang W,et al.Identification of Lonicera japoni-ca by PCR-RFLP and allele-specific diagnostic PCR based on se-quences of internal transcribed spacer regions[J].Planta Med,2010,76(5):497.
- [9]Sun Z,Gao T,Yao H,et al.Identification of Lonicera japonicaand its related species using the DNA barcoding method[J].Planta Med,2011,77(3):301.
- [10]蒋超,袁媛,刘贵明,等.基于EST-SSR的金银花分子鉴别方法研究[J].药学学报,47(2):803.
- [11]Wang H,Kim M K,Kwon W S,et al.Molecular authenticationof Panax ginseng and ginseng products using robust SNP markersin ribosomal external transcribed spacer region[J].J Pharm Bi-omed Anal,2011,55(5):972.
- [12]Wang H,Kim M K,Kim Y J,et al.Molecular authentication ofthe oriental medicines Pericarpium Citri Reticulatae and Citri Un-shius Pericarpium using SNP markers[J].Gene,2012,494(1):92.
- [13]Jigden B,Wang H,Kyum K M,et al.Authentication of the ori-ental medicinal plant Ligusticum tenuissimum(Nakai)Kitagawa(Korean Go-Bon)by multiplex PCR[J].Planta Med,2010,76(6):648.
- [14]Liu Q,Thorland E C,Heit J A,et al.Overlapping PCR for bidi-rectional PCR amplification of specific alleles:a rapid one-tubemethod for simultaneously differentiating homozygotes and hetero-zygotes[J].Genome Res,1997,7(4):389.
- [15]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,2008,5(6):524.
- [16]王柯,章金涛,运玉霞,等.PCR-CTPP:一种基于错配技术的SNP分型方法的改良[J].遗传,2011,33(2):182.
- [17]卜莹,古卓良,张晓丹,等.四引物PCR扩增反应的单管SNP快速测定法[J].中国生物化学与分子生物学报,2004,20(2):252.
- 双向位点特异性PCR
- 金银花
- 分子鉴定
bidirectional PCR amplification of specific alleles - Lonicera japonica
- molecular authentication
- 蒋超
- 张雅华
- 陈敏
- 袁媛
- 林淑芳
- 吴志刚
JIANG Chao- ZHANG Ya-hua
- CHEN Min
- YUAN Yuan*
- LIN Shu-fang
- WU Zhi-gang(Institute of Chinese Materia Medica
- China Academy of Chinese Medical Sciences
- Beijing 100700
- China)
- 蒋超
- 张雅华
- 陈敏
- 袁媛
- 林淑芳
- 吴志刚
JIANG Chao- ZHANG Ya-hua
- CHEN Min
- YUAN Yuan*
- LIN Shu-fang
- WU Zhi-gang(Institute of Chinese Materia Medica
- China Academy of Chinese Medical Sciences
- Beijing 100700
- China)