基于熔解曲线峰形特异性鉴别川贝母真伪
|
摘要
目的探讨基于熔解曲线峰形及退火温度(T_m值)在川贝母真伪鉴别中的应用。方法采用《中国药典》2015年版聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)鉴别法检测川贝母样品。使用CLUSTAL X软件比对NCBI-Genbank数据库下载的川贝母及其常见伪品特征序列,设计并验证引物与实时荧光PCR,检测市售样品。结果川贝母熔解曲线呈尖锐峰且T_m值为80.98℃,与常见伪品峰形有明显区别。结论此研究利用熔解曲线峰形特异性鉴定川贝母,实验操作简单,鉴定结果直观。
Objective To explore the application of Real-Time PCR(RT-PCR)DNA melting curve peak shape and its annealing temperature(T_m value)in identifying Fritillaria cirrhosa bulbus. Methods DNA samples were detected using PCR-RFLP method referring to Chinese Pharmacopoeia. DNA sequences of Fritillaria cirrhosa bulbus and its common pseudo-products from the NCBI-Genbank database were collected and compared by CLUSTAL software.Suitable primers were designed and verified. The melting-curve PCR method verified by reference medicine were used to detect DNA of commercial samples. Results Fritillaria cirrhosa bulbus can be identified by a single sharp peak at the annealing temperature of 80.98 ℃,that is significantly different from those of pseudo-products. Conclusion The study suggests that the method of RT-PCR with DNA melting curve peak shape analysis is convenient and intuitive for detecting Fritillaria cirrhosa bulbus.
Objective To explore the application of Real-Time PCR(RT-PCR)DNA melting curve peak shape and its annealing temperature(T_m value)in identifying Fritillaria cirrhosa bulbus. Methods DNA samples were detected using PCR-RFLP method referring to Chinese Pharmacopoeia. DNA sequences of Fritillaria cirrhosa bulbus and its common pseudo-products from the NCBI-Genbank database were collected and compared by CLUSTAL software.Suitable primers were designed and verified. The melting-curve PCR method verified by reference medicine were used to detect DNA of commercial samples. Results Fritillaria cirrhosa bulbus can be identified by a single sharp peak at the annealing temperature of 80.98 ℃,that is significantly different from those of pseudo-products. Conclusion The study suggests that the method of RT-PCR with DNA melting curve peak shape analysis is convenient and intuitive for detecting Fritillaria cirrhosa bulbus.
引文
[1]国家药典委员会.中华人民共和国药典(一部)[S].北京:中国医药科技出版社,2015:36-38.
[2]赵文龙,陈红刚,林丽,等.不同基原的中药川贝母生境适宜性分布[J].生态学杂志,2018,37(4):1037-1042.
[3]高梓童,王晓玥,刘杨,等.基于ITS2序列的川贝母中成药的鉴定[J].中国科学(生命科学),2018,48(4):482-489.
[4]肖培根,姜艳,李萍,等.中药贝母的基原植物和药用亲缘学的研究[J].植物分类学报,2007,45(4):473-487.
[5]张翔,李文涛,段宝忠,等.基于品质特征的贝母类药材品种分类研究[J].中草药,2018,49(9):2140-2146.
[6]胡伟,陈伟盛,林秀旎,等.聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)鉴定川贝母药材的方法优化[J].药物分析杂志,2017,37(9):1716-1720.
[7]张文娟,刘薇,魏锋,等.聚合酶链式反应-限制性片段长度多态性法用于检定川贝母掺伪情况的研究[J].药物分析杂志,2014,34(10):1830-1835.
[8]徐传林,李会军,李萍,等.川贝母药材分析鉴定方法研究[J].中国药科大学学报,2010,41(3):226-230.
[9]赵仲麟,常志远,袁超,等.PCR-RFLP定量检测川贝母真伪的研究[J].河南农业大学学报,2018,52(02):249-253.
[10]李庆,陈新,王曙.川贝母复合群之间的分子生物学亲缘关系的探讨[J].华西药学杂志,2010,25(2):140-143.
[11]黎开强,吴卫,郑有良,等.川产贝母种质资源遗传多样性的ISSR分析[J].中国中药杂志,2009,34(17):2149-2154.
[12]罗达龙,黄林杰,黄琳.实时荧光定量PCR对川贝母的鉴别应用[J].中国药师,2016,19(6):1068-1070.
[13]凌睿,薛建丽,杨军,等.Real-Time PCR熔解曲线及Myostatin基因在肉类掺假快速鉴别中的应用[J].食品科技,2013,38(5):318-322.
[14]陈虞超,郭生虎,关雅静,等.贝母属药用植物研究进展[J/OL].分子植物育种,[2018-11-20].http://kns.cnki.net/kcms/detail/46.1068.S.20181119.1335.004.html
[15]安钢力.实时荧光定量PCR技术的原理及其应用[J].中国现代教育装备,2018,30(1):19-21.
[16]CHEN H X,ZHANG X,HOU H T,et al.Identication of a novel and functional mutation in the TBX5 gene in a patient by screening from 354 patients with isolated ventricular septal defect[J].European Journal of Medical Genetics,2017,60(7):385-390.
[17]MISHRA P,KUMAR A,NAGIREDDY A,et al.DNA barcoding:an efficient tool to overcome authentication challenges in the herbal market[J].Plant Biotechnology Journal,2015,14(1):8-21.
[18]王丽丽.DNA条形码鉴定中药材和中成药的应用研究[D].北京:北京协和医学院,2016.
[2]赵文龙,陈红刚,林丽,等.不同基原的中药川贝母生境适宜性分布[J].生态学杂志,2018,37(4):1037-1042.
[3]高梓童,王晓玥,刘杨,等.基于ITS2序列的川贝母中成药的鉴定[J].中国科学(生命科学),2018,48(4):482-489.
[4]肖培根,姜艳,李萍,等.中药贝母的基原植物和药用亲缘学的研究[J].植物分类学报,2007,45(4):473-487.
[5]张翔,李文涛,段宝忠,等.基于品质特征的贝母类药材品种分类研究[J].中草药,2018,49(9):2140-2146.
[6]胡伟,陈伟盛,林秀旎,等.聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)鉴定川贝母药材的方法优化[J].药物分析杂志,2017,37(9):1716-1720.
[7]张文娟,刘薇,魏锋,等.聚合酶链式反应-限制性片段长度多态性法用于检定川贝母掺伪情况的研究[J].药物分析杂志,2014,34(10):1830-1835.
[8]徐传林,李会军,李萍,等.川贝母药材分析鉴定方法研究[J].中国药科大学学报,2010,41(3):226-230.
[9]赵仲麟,常志远,袁超,等.PCR-RFLP定量检测川贝母真伪的研究[J].河南农业大学学报,2018,52(02):249-253.
[10]李庆,陈新,王曙.川贝母复合群之间的分子生物学亲缘关系的探讨[J].华西药学杂志,2010,25(2):140-143.
[11]黎开强,吴卫,郑有良,等.川产贝母种质资源遗传多样性的ISSR分析[J].中国中药杂志,2009,34(17):2149-2154.
[12]罗达龙,黄林杰,黄琳.实时荧光定量PCR对川贝母的鉴别应用[J].中国药师,2016,19(6):1068-1070.
[13]凌睿,薛建丽,杨军,等.Real-Time PCR熔解曲线及Myostatin基因在肉类掺假快速鉴别中的应用[J].食品科技,2013,38(5):318-322.
[14]陈虞超,郭生虎,关雅静,等.贝母属药用植物研究进展[J/OL].分子植物育种,[2018-11-20].http://kns.cnki.net/kcms/detail/46.1068.S.20181119.1335.004.html
[15]安钢力.实时荧光定量PCR技术的原理及其应用[J].中国现代教育装备,2018,30(1):19-21.
[16]CHEN H X,ZHANG X,HOU H T,et al.Identication of a novel and functional mutation in the TBX5 gene in a patient by screening from 354 patients with isolated ventricular septal defect[J].European Journal of Medical Genetics,2017,60(7):385-390.
[17]MISHRA P,KUMAR A,NAGIREDDY A,et al.DNA barcoding:an efficient tool to overcome authentication challenges in the herbal market[J].Plant Biotechnology Journal,2015,14(1):8-21.
[18]王丽丽.DNA条形码鉴定中药材和中成药的应用研究[D].北京:北京协和医学院,2016.