杂交探针技术结合熔解曲线鉴别川贝母与伊贝母的研究
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摘要
目的建立一种快速、准确鉴别川贝母和伊贝母的方法。方法针对川贝母与伊贝母的ITS1基因特异性位点设计杂交探针,采用聚合酶链反应(Polymerase Chain Reaction,PCR)扩增特异性片段,将其与探针杂交后,引发荧光共振能量转移,通过实时PCR仪绘制目标DNA片段扩增过程中的熔解曲线,根据熔解温度(Melting Temperature,T_m)对川贝母正伪品进行分析。结果两种贝母的熔解曲线、T_m具有特异性,通过该方法可以有效鉴别出川贝母和伊贝母,模版DNA最低检出限达到了0.2 pg,具有较高的灵敏度。结论该技术可用于川贝母质量控制,可作为《中国药典》方法的有效补充,对其他疑难品种的鉴别具有借鉴作用。
Objective To establish a rapid and accurate method for identifying Fritillaria cirrhosa and Fritillaria pallidiflora. Methods In this study,a hybridization probe was designed for the specific site of the ITS1 gene of F.cirrhosa. The specific fragment was amplified by Polymerase Chain Reaction(PCR) and hybridized with probes toinitiate fluorescence resonance energy transfer. The melting curve of the amplified target DNA fragment was drawn byreal-time PCR instrument,and identified by Melting Temperature(T_m). Results The melting curves and T_m valuesof the two kinds of Fritillaria are specific,that by this method,F. cirrhosa and F. pallidiflora can be effectivelyidentified. The minimum detection limit of template DNA is 0.2 pg,showing a high sensitivity. Conclusion Thistechnology can be used for the quality control of F. cirrhosa,as an effective supplement to Chinese Pharmacopoeia,and has great potential for the identification of other difficult varieties.
Objective To establish a rapid and accurate method for identifying Fritillaria cirrhosa and Fritillaria pallidiflora. Methods In this study,a hybridization probe was designed for the specific site of the ITS1 gene of F.cirrhosa. The specific fragment was amplified by Polymerase Chain Reaction(PCR) and hybridized with probes toinitiate fluorescence resonance energy transfer. The melting curve of the amplified target DNA fragment was drawn byreal-time PCR instrument,and identified by Melting Temperature(T_m). Results The melting curves and T_m valuesof the two kinds of Fritillaria are specific,that by this method,F. cirrhosa and F. pallidiflora can be effectivelyidentified. The minimum detection limit of template DNA is 0.2 pg,showing a high sensitivity. Conclusion Thistechnology can be used for the quality control of F. cirrhosa,as an effective supplement to Chinese Pharmacopoeia,and has great potential for the identification of other difficult varieties.
引文
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[2]张文娟,尚柯,魏锋,等.聚合酶链式反应-限制性片段长度多态性方法对太白贝母鉴别检验的适用性探讨[J].中国现代中药,2015,17(9):905-910.
[3]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.
[4]王聪,王曙,马静.川贝母的HPLC指纹图谱研究[J].华西药学杂志,2010,25(1):61-63.
[5]王琳玲,王玲玲,于国强,等.川贝母的HPLC指纹图谱研究[J].华西药学杂志,2016,31(5):497-501.
[6]雷艳辉,李会军,李萍.川贝母药材生物碱成分HPLC-ELSD特征图谱的研究[J].中成药,2014,36(7):1477-1481.
[7]罗焜,马培,姚辉,等.基于ITS2序列鉴定川贝母及其混伪品基原植物[J].世界科学技术-中医药现代化,2012,14(1):1153-1158.
[8]徐传林,李会军,李萍,等.川贝母药材分子鉴定方法研究[J].中国药科大学学报,2010,41(3):226-230.
[9]XIN G Z,LAM Y C,MAIWULANJIANG M,et al.Authentication of bulbus fritillariae cirrhosae by RAPD-derived DNA markers[J].Molecules,2014,19(3):3450-3459.
[10]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.
[11]王倩倩,谭勇,张芳,等.伊犁贝母的生药学鉴定[J].中国药房,2012,23(43):4089-4091.
[12]国家药典委员会.中国药典(一部)[M].北京:中国医药科技出版社,2010:83
[13]HIGUCHI R,DOLLINGER G,WALSH P S,et al.Simultaneous amplification and detection of specific DNA sequences[J].Biotechnology(NY),1992,10:413.