何首乌等位基因特异性PCR鉴别方法研究
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摘要
目的建立一种快速鉴别何首乌真伪的方法。方法通过何首乌及其混伪品的psb A-trn H基因序列,寻找SNP位点并设计特异性引物,对来自于不同产地的何首乌及其3个同属混伪品进行PCR扩增,优化反应体系条件,并对此方法进行考察。结果建立了何首乌特异性PCR的方法,在退火温度48℃、循环次数30时仅有何首乌能扩增得到191 bp的特异性条带,伪品则无。结论等位基因特异性PCR鉴别何首乌真伪方法简单、可靠。
Objective To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods Based on psb A-trn H sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F.multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative.Conclusion It's simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.
Objective To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods Based on psb A-trn H sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F.multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative.Conclusion It's simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.
引文
[1]唐慎微.重修政和经史证类备用本草[M].北京:人民卫生出版社,1957.
[2]中国药典[S].一部.2015.
[3]任红微,魏静,高秀梅,等.何首乌及其主要化学成分药理作用及机制研究进展[J].药物评价研究,2018,41(7):1357-1362.
[4]谢宗万.中药材品种论述[M].上海:上海科学技术出版社,1990.
[5]葛李薇,武智聪,张勉,等.何首乌及其同属混伪品的鉴别研究[J].中国野生植物资源,2011,30(3):64-68.
[6]刘越,罗定强,王国海,等.何首乌药材及常见伪品的真伪鉴别方法研究[J].中药材,2017,40(6):1305-1308.
[7]Yan H J,Fang Z J,Zhang H Y,et al.Fallopia multiflora var.angulata,a new combination in the Polygonaceae from China[J].Novon,2011,21:388-391.
[8]朱华,黄权芳,周燕华,等.HPLC法测定广西不同产地何首乌中二苯乙烯苷的含量[J].广西中医药,2006,29(6):53-54.
[9]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):188-192.
[10]蒋超,罗宇琴,袁媛,等.多重位点特异性PCR鉴别人参、三七、西洋参掺杂[J].中国中药杂志,2017,42(7):1319-1323.
[11]魏艺聪,袁媛,陈建雄,等.快速PCR法鉴别鱼腥草与百部还魂的方法研究[J].中草药,2016,47(12):2163-2166.
[12]李敏,黄龙妹,赵欣,等.浙贝母特异性PCR鉴定方法研究[J].中草药,2014,45(12):1754-1757.
[13]黎洁文.基于ITS2序列分析的何首乌PCR-RFLP和AS-PCR的分子鉴别研究[D].广州:广州中医药大学,2015.
[14]严萍,赵树进.中药何首乌总基因组DNA的提取[J].时珍国医国药,2007,18(3):607-609.
[15]白明明,孙小芹,郭建林,等.基于psb A-trn H分析的何首乌野生居群遗传多样性[J].植物资源与环境学报,2012,21(2):36-44.
[16]Wang H T,Kim M K,Kim Y J,et al.Molecular authentication of the oriental medicines Pericarpium Citri Reticulatae and Citri Unshius Pericarpium using SNP markers[J].Gene,2012,494(2012):92-95.
[17]马厚勋,牛永红,李章勇,等.等位基因特异性引物PCR技术及其应用研究[J].生物技术,2005,15(1):15-18.
[18]陈琳玲,张佳,彭翠英,等.DNA聚合酶高保真机理的新发现及其在SNP分析中的应用[J].遗传,2005,27(2):279-283.
[19]郑传进,生书晶,赵树进.基于trnL-trnF序列分析的何首乌PCR-RFLP分子鉴别[J].中药材,2012,35(4):543-547.
[20]呙军,赵树进,郑传进.何首乌及其混伪品序列相关扩增多态性反应体系的优化[J].时珍国医国药,2009,20(5):1137-1139.
[21]杨慧洁,杨世海,张淑丽,等.药用植物DNA条形码研究进展[J].中草药,2014,45(18):2581-2587.
[22]张宏意,袁林林,饶秋红,等.不同产地何首乌叶绿体psb A-trn H基因序列分析[J].中草药,2018,49(5):1146-1149.
[23]刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度不当导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-114.
[2]中国药典[S].一部.2015.
[3]任红微,魏静,高秀梅,等.何首乌及其主要化学成分药理作用及机制研究进展[J].药物评价研究,2018,41(7):1357-1362.
[4]谢宗万.中药材品种论述[M].上海:上海科学技术出版社,1990.
[5]葛李薇,武智聪,张勉,等.何首乌及其同属混伪品的鉴别研究[J].中国野生植物资源,2011,30(3):64-68.
[6]刘越,罗定强,王国海,等.何首乌药材及常见伪品的真伪鉴别方法研究[J].中药材,2017,40(6):1305-1308.
[7]Yan H J,Fang Z J,Zhang H Y,et al.Fallopia multiflora var.angulata,a new combination in the Polygonaceae from China[J].Novon,2011,21:388-391.
[8]朱华,黄权芳,周燕华,等.HPLC法测定广西不同产地何首乌中二苯乙烯苷的含量[J].广西中医药,2006,29(6):53-54.
[9]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):188-192.
[10]蒋超,罗宇琴,袁媛,等.多重位点特异性PCR鉴别人参、三七、西洋参掺杂[J].中国中药杂志,2017,42(7):1319-1323.
[11]魏艺聪,袁媛,陈建雄,等.快速PCR法鉴别鱼腥草与百部还魂的方法研究[J].中草药,2016,47(12):2163-2166.
[12]李敏,黄龙妹,赵欣,等.浙贝母特异性PCR鉴定方法研究[J].中草药,2014,45(12):1754-1757.
[13]黎洁文.基于ITS2序列分析的何首乌PCR-RFLP和AS-PCR的分子鉴别研究[D].广州:广州中医药大学,2015.
[14]严萍,赵树进.中药何首乌总基因组DNA的提取[J].时珍国医国药,2007,18(3):607-609.
[15]白明明,孙小芹,郭建林,等.基于psb A-trn H分析的何首乌野生居群遗传多样性[J].植物资源与环境学报,2012,21(2):36-44.
[16]Wang H T,Kim M K,Kim Y J,et al.Molecular authentication of the oriental medicines Pericarpium Citri Reticulatae and Citri Unshius Pericarpium using SNP markers[J].Gene,2012,494(2012):92-95.
[17]马厚勋,牛永红,李章勇,等.等位基因特异性引物PCR技术及其应用研究[J].生物技术,2005,15(1):15-18.
[18]陈琳玲,张佳,彭翠英,等.DNA聚合酶高保真机理的新发现及其在SNP分析中的应用[J].遗传,2005,27(2):279-283.
[19]郑传进,生书晶,赵树进.基于trnL-trnF序列分析的何首乌PCR-RFLP分子鉴别[J].中药材,2012,35(4):543-547.
[20]呙军,赵树进,郑传进.何首乌及其混伪品序列相关扩增多态性反应体系的优化[J].时珍国医国药,2009,20(5):1137-1139.
[21]杨慧洁,杨世海,张淑丽,等.药用植物DNA条形码研究进展[J].中草药,2014,45(18):2581-2587.
[22]张宏意,袁林林,饶秋红,等.不同产地何首乌叶绿体psb A-trn H基因序列分析[J].中草药,2018,49(5):1146-1149.
[23]刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度不当导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-114.