基于LAMP的中药材川贝母真伪鉴别方法的建立
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摘要
目的:基于荧光-环状等温扩增技术,建立川贝母快速检测方法,实现中药材川贝母的荧光可视化快速鉴定。方法:依据川贝母物种特异性序列设计5套LAMP引物,并测试方法特异性和灵敏度。结果:引物Set1扩增效率最高;使用该引物建立的川贝母LAMP检测方法仅能够在6种川贝母基源药材中扩增出扩增曲线和荧光变化,在伊贝母、浙贝母、平贝母等7种常见混淆品中无扩增产物,方法特异性良好;方法检测灵敏度为0.1%。结论:本研究建立的基于LAMP的川贝母鉴定方法具有较好的特异性和灵敏度,其快速、操作简单、易观察的优点,填补了中药材川贝母真伪快速筛查的技术空白。
Objective:To establish rapid visual identification method for Fritillaria cirrhosa based on loop-mediated isothermal amplification. Methods:According to the DNA sequence of the target,five sets of primers were designed and screened by amplification efficiency. Specificity and sensitivity of the method was validated. Results:The LAMP detection method established by using primer set 1 showed best performance on amplification efficiency,specificity and sensitivity. Turbidity and fluorescence changes could be observed in 6 kinds of Fritillariae,and there was no amplification product in 7 common adulterants. The sensitivity of the LAMP method was 0.1%.Conclusion:The proposed LAMP method is specific,sensitive,accurate and simple and easy to observe,which fills the gap for the authentication of Fritillaria cirrhosa.
Objective:To establish rapid visual identification method for Fritillaria cirrhosa based on loop-mediated isothermal amplification. Methods:According to the DNA sequence of the target,five sets of primers were designed and screened by amplification efficiency. Specificity and sensitivity of the method was validated. Results:The LAMP detection method established by using primer set 1 showed best performance on amplification efficiency,specificity and sensitivity. Turbidity and fluorescence changes could be observed in 6 kinds of Fritillariae,and there was no amplification product in 7 common adulterants. The sensitivity of the LAMP method was 0.1%.Conclusion:The proposed LAMP method is specific,sensitive,accurate and simple and easy to observe,which fills the gap for the authentication of Fritillaria cirrhosa.
引文
[1]陈杰,吕银姣.川贝母及其混淆品东贝母的鉴别[J].时珍国医国药,2000,11(9):807CHEN J,LU YJ.Identification of Fritillaria and its adulterants[J].Lishizhen Med Mate Med Res,2000,11(9):807
[2]吴玉良,杨杰,吴纯洁.川贝母综合利用的思考[C].中华中医药学会中药炮制分会2011年学术年会论文集2011WU YL,YANG J,WU CJ.Consideration of Comprehensive Utilization of Fritillaria[C].Collected Papers of Academic Annual Conference of Traditional Chinese Medicine,2011
[3]王保华.川贝母、浙贝母、平贝母的异同[J].中医临床研究,2015(23):23WANG BH.The similarities and differences of Chuanbeimu,Zhebeimu and Pingbeimu[J].Clin J Chin Med,2015(23):23
[4]吴小婷.川贝母的真伪鉴别[J].海峡药学,2014(6):39WU XT.Identification of Fritillary bulb[J].Strait Pharm J,2014(6):39
[5]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63
[6]YANO A,ISHIMARU R,HUJIKATA R.Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification[J].Microbiol Methods,2007,68(2):414
[7]NAGAMINE K,WATANABE K,OHTSUKA K,et al.Loopmediated isothermal amplification reaction using a nondenatured template[J].Clin Chem,2001,47(9):1742
[8]NAGAMINE K,HASE T,NOTOMI T.Accelerated reaction by loopmediated isothermal amplification using loop primers[J].Mol Cell Probes,2002,16(3):223
[9]MORI Y,KITAO M,TOMITA N,et al.Real-time turbidimetry of LAMP reaction for quantifying template DNA[J].J Biochem Biophys Methods,2004,59(2):145
[10]MORI Y,HIRANO T,NOTOMI T.Sequence specific visual detection of LAMP reactions by addition of cationic polymers[J].BMC Biotechnol,2006,6:3
[11]IHIRA M,OHTA A,SUGATA K,et al.Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B[J].J Virol Methods,2008,154(1/2):223
[12]CURTIS KA,RUDOLPH DL,OWEN SM.Rapid detection of HIV-1 by reverse transcription,loop-mediated isothermal amplification(RT-LAMP)[J].J Virol Methods,2008,151(2):264
[13]GAO HW,LEI ZV,JIA JT,et al.Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat[J].J Microbiol Methods,2008,77(2):198
[14]XIN GZ,LAM YC,MAIWULANJIANG M,et al.Authentication of Fritillariae cirrhosae Bulbus by RAPD-derived DNA markers[J].Molecules,2014,19(3):3450
[15]潘莉,徐道华.川贝母的真伪鉴别[J].中国药业,2008,17(20):55PAN L,XU DH.Identification of Fritillary bulb[J].China Pharm,2008,17(20):55
[16]刘志梅,赵华,明延波.川贝母及其伪品小东贝母的鉴别[J].中国当代医药,2013.20(8):70-71.LIU ZM,ZHAO H,MING YB.Identification of Fritillariae cirrhosae Bulbus and its adulterant-small east Fritillaria[J].China Mod Med,2013.20(8):70
[17]王文娜,陈地灵,朱梅芳,等.激光拉曼光谱法无损分析鉴别川贝母[J].光谱学与光谱分析,2013,33(8):2109WANG WN,CHEN DL,ZHU MF,et al.The analysis and identification of Fritillaria Cirrhosa by Raman spectra[J].Spectrosc Spect Anal,2013,33(8):2109
[18]中华人民共和国药典2015年版.一部[S].2015:36ChP 2015.VolⅠ[S].2015:36
[19]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):1068LUO DL,HUANG LJ,HUANG L.Application of real-time fluorescent quantitative PCR in the identification of Fritillariae Cirrhosae Bulbus[J].China Pharm,2016,19(6):1068
[20]ZOU Y,MASON MG,WANG Y,et al.Nucleic acid purification from plants,animals and microbes in under 30 seconds[J].PLoSBiol,2017,15(11):e2003916
[2]吴玉良,杨杰,吴纯洁.川贝母综合利用的思考[C].中华中医药学会中药炮制分会2011年学术年会论文集2011WU YL,YANG J,WU CJ.Consideration of Comprehensive Utilization of Fritillaria[C].Collected Papers of Academic Annual Conference of Traditional Chinese Medicine,2011
[3]王保华.川贝母、浙贝母、平贝母的异同[J].中医临床研究,2015(23):23WANG BH.The similarities and differences of Chuanbeimu,Zhebeimu and Pingbeimu[J].Clin J Chin Med,2015(23):23
[4]吴小婷.川贝母的真伪鉴别[J].海峡药学,2014(6):39WU XT.Identification of Fritillary bulb[J].Strait Pharm J,2014(6):39
[5]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63
[6]YANO A,ISHIMARU R,HUJIKATA R.Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by loop-mediated isothermal amplification[J].Microbiol Methods,2007,68(2):414
[7]NAGAMINE K,WATANABE K,OHTSUKA K,et al.Loopmediated isothermal amplification reaction using a nondenatured template[J].Clin Chem,2001,47(9):1742
[8]NAGAMINE K,HASE T,NOTOMI T.Accelerated reaction by loopmediated isothermal amplification using loop primers[J].Mol Cell Probes,2002,16(3):223
[9]MORI Y,KITAO M,TOMITA N,et al.Real-time turbidimetry of LAMP reaction for quantifying template DNA[J].J Biochem Biophys Methods,2004,59(2):145
[10]MORI Y,HIRANO T,NOTOMI T.Sequence specific visual detection of LAMP reactions by addition of cationic polymers[J].BMC Biotechnol,2006,6:3
[11]IHIRA M,OHTA A,SUGATA K,et al.Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B[J].J Virol Methods,2008,154(1/2):223
[12]CURTIS KA,RUDOLPH DL,OWEN SM.Rapid detection of HIV-1 by reverse transcription,loop-mediated isothermal amplification(RT-LAMP)[J].J Virol Methods,2008,151(2):264
[13]GAO HW,LEI ZV,JIA JT,et al.Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat[J].J Microbiol Methods,2008,77(2):198
[14]XIN GZ,LAM YC,MAIWULANJIANG M,et al.Authentication of Fritillariae cirrhosae Bulbus by RAPD-derived DNA markers[J].Molecules,2014,19(3):3450
[15]潘莉,徐道华.川贝母的真伪鉴别[J].中国药业,2008,17(20):55PAN L,XU DH.Identification of Fritillary bulb[J].China Pharm,2008,17(20):55
[16]刘志梅,赵华,明延波.川贝母及其伪品小东贝母的鉴别[J].中国当代医药,2013.20(8):70-71.LIU ZM,ZHAO H,MING YB.Identification of Fritillariae cirrhosae Bulbus and its adulterant-small east Fritillaria[J].China Mod Med,2013.20(8):70
[17]王文娜,陈地灵,朱梅芳,等.激光拉曼光谱法无损分析鉴别川贝母[J].光谱学与光谱分析,2013,33(8):2109WANG WN,CHEN DL,ZHU MF,et al.The analysis and identification of Fritillaria Cirrhosa by Raman spectra[J].Spectrosc Spect Anal,2013,33(8):2109
[18]中华人民共和国药典2015年版.一部[S].2015:36ChP 2015.VolⅠ[S].2015:36
[19]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):1068LUO DL,HUANG LJ,HUANG L.Application of real-time fluorescent quantitative PCR in the identification of Fritillariae Cirrhosae Bulbus[J].China Pharm,2016,19(6):1068
[20]ZOU Y,MASON MG,WANG Y,et al.Nucleic acid purification from plants,animals and microbes in under 30 seconds[J].PLoSBiol,2017,15(11):e2003916