三七特异性PCR真伪鉴别方法研究

目的:建立一种用于鉴别三七的特异性PCR鉴别方法。方法:通过比较三七及其混伪品的psbA-trnH基因序列差异,根据变异位点设计三七的特异性鉴别引物,优化反应体系,并对此方法进行耐受性和适用性的考察和验证。结果:构建了鉴别三七特异性PCR方法,在47 ℃的退火温度下,用鉴别引物对所有样品进行扩增,仅三七正品能扩增得到约241 bp的特异性条带,其他药材均为阴性扩增。结论:该鉴别引物能准确、特异性地鉴别三七,可用于三七药材的鉴定。

Objective:To establish a specificity PCR method for identification of Panax notoginseng.Methods:Based on the difference of psbA-trnH gene DNA sequences among Panax notoginseng and its adulterants,the specific primer was designed,the reaction conditions were optimized,and the PCR method for identification of Panax notoginseng was explored and verified in terms of tolerance and feasibility.Results:Panax notoginseng PCR identification system was constructed.The annealing temperature was 47 ℃ and the 241 bp fragment was only amplified from DNA templates of Panax notoginseng;nevertheless its adulterants were negative.Conclusion:The method can be used to identify traditional Chinese medicines Panax notoginseng accurately and specifically.

[1] 中国植物志编辑委员会.中国植物志[M].北京:科学出版社,1978:179 Editorial Committee of Flora of China.Flora Reipublicae Popularis Sinicae[M].Beijing:Science Press,1978:179
[2] NG TB.Pharmacological activity of sanchi ginseng(Panax notoginseng)[J].J Pharm Pharmacol,2006,58(8):1007
[3] 崔秀明,黄璐琦,郭兰萍,等.中国三七产业现状及发展对策[J].中国中药杂志,2014,39(4):553 CUI XM,HUANG LQ,GOU LP,et al.Chinese Sanqi industry status and development countermeasures[J].China J Chin Mater Med,2014,39(4):553
[4] 董晶晶,陈娟,戈延茹,等.激光拉曼光谱法无损鉴别三七及其伪品[J].激光与光电子学进展,2014,51(5):204 DONG JJ,CHEN J,GE YR,et al.Nondestructive identification of Panax notoginseng and its analogues via laser Raman spectroscopy[J].Laser Optoelectron Prog,2014,51(5):204
[5] 崔翰明,张秋燕,彭亮,等.三七不同部位及总皂苷提取物HPLC指纹图谱研究[J].中药材,2011,34(3):362 CUI HM,ZHANG QY,PENG L,et al.Study on HPLC fingerprint of Panax notoginseng different parts and total saponins extract[J].J Chin Med Mater,2011,34(3):362
[6] 张南平,张文娟,魏锋,等.中药DNA分子鉴别标准研究方法[J].药物分析杂志,2014,34(11):2066 ZHANG NP,ZHANG WJ,WEI F,et al.The standard method of DNA molecular identification for traditional Chinese medicine[J].Chin J Pharm Anal,2014,34(11):2066
[7] 石林春,陈俊,向丽,等.基于ITS2条形码的中药材天南星及其混伪品DNA分子鉴定[J].中国中药杂志,2014,39(12):2176 SHI LC,CHEN J,XIANG L,et al.DNA barcoding identification between Arisaematis Rhizoma and its adulterants based on ITS2 sequences[J].China J Chin Mater Med,2014,39(12):2176
[8] 崔占虎,龙平,王颖莉,等.DNA分子标记技术在中成药鉴定中的应用与展望[J].中药材,2015,38(1):188 CUI ZH,LONG P,WANG YL,et al.Application and prospect of DNA molecular markers in the identification of Chinese medicine[J].J Chin Med Mater,2015,38(1):188
[9] 中国药典2010年版.一部[S].2010:72,349 ChP 2010.Vol Ⅰ[S].2010:72,349
[10] 詹鑫婕,田程,张媛,等.基于ITS2条形码SNPs的人参和西洋参PCR-SSCP分子鉴别研究[J].中国中药杂志,2012,37(24):3748 ZHAN XJ,TIAN C,ZHANG Y,et al.PCR-SSCP molecular identification of Panax ginseng and P.quinquefolius based on ITS2 barcoding SNPs[J].China J Chin Mater Med,2012,37(24):3748
[11] 曹亮,秦双双,袁媛,等.薄荷、留兰香特异性引物PCR鉴别方法研究[J].中药材,2014,37(1):41 CAO L,QIN SS,YUAN Y,et al.Molecular identification of Mentha haplocalyx and Mentha spicata with specific primers multi-PCR system[J].J Chin Med Mater,2014,37(1):41
[12] 曹晖,刘玉萍,伏见裕利,等.三七及其伪品的DNA测序鉴别[J].中药材,2001,24(6):398 CAO H,LIU YP,FUSHIMI H,et al.Panax notoginseng and DNA sequencing of its pseudo products[J].J Chin Med Mater,2001,24(6):398
[13] 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 CHEN SL,YAO H,HAN JP,et al.Traditional Chinese medicine DNA barcoding molecular identification principle[J].China J Chin Mater Med,2013,38(2):141