多重连接探针扩增技术检测川贝母掺伪的研究
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摘要
目的:着重探讨采用新技术检测川贝母的真伪。方法:本研究利用多重连接探针扩增(MLPA)技术特异、敏感等技术优势,以川贝母、平贝母、伊贝母、浙贝母和湖北贝母为研究对象,针对川贝母及非川贝母ITS1序列设计了10条特异探针,建立川贝母掺伪检测及相对定量的MLPA扩增方法。结果:针对探针混合物分别以川贝母及4种非川贝母对照药材为模板,从单个样品中只能扩增出单一扩增峰,表明了探针高特异性。敏感性分析结果表明,MLPA扩增最低检测限度可达到掺伪10%,并且目的扩增峰面积比与掺伪比例基本一致。检测市场抽查样品验证了MLPA扩增方法在川贝母掺伪检测中的适用性,表明该方法可有效检出川贝母药材掺伪,并可反映市场药材掺伪比例。结论:本研究基于MLPA建立的川贝母掺伪检测方法,具有特异性强及敏感性高的特点,可为川贝母药材检验监管提供技术支持,为中药材掺伪检测技术的建立提供参考。
Objective:Current detection technologies for Fritillariae Cirrhosae Bulbus analysis is mostly targeted at true or false identification rather than adulteration.In view of the serious adulteration of this medicine,the study of new technology has been a focus of attention.Methods:Multi-parameter assay based on multiplex ligation-dependent probe amplification(MLPA)technology with the advantages of specificity and sensitivity was used.Ten specific probes targeted at Fritillariae Cirrhosae Bulbus,F.Ussuriensis Bulbus,F.Pallidiflorae Bulbus,F.Thunbergii Bulbus and F.Hupehensis Bulbus,were designed respectively and the relative quantitative method for adulteration was established.Results:The mixture of the ten probes only amplified the specific amplification peak from these five Bulbus Fritillariae templates,indicating the probes had good specificity.The result of sensitivity test showed that the adulteration ratio of F.Cirrhosae Bulbus in one MLPA reaction was up to 10%,and the area ratio of the target amplification peaks was in accordance with the adulteration ratio.Then the adulteration test applicability of F.Cirrhosae Bulbus using MLPA method was verified by testing the samples from the market,and the adulteration ratio of the market-collected F.Cirrhosae Bulbus samples was also reflected.Conclusion:The MLPA method described in this article can effectively detect the adulteration in F.Cirrhosae Bulbus,which provids a technical support for inspection and supervision of this important traditional Chinese medicine.
Objective:Current detection technologies for Fritillariae Cirrhosae Bulbus analysis is mostly targeted at true or false identification rather than adulteration.In view of the serious adulteration of this medicine,the study of new technology has been a focus of attention.Methods:Multi-parameter assay based on multiplex ligation-dependent probe amplification(MLPA)technology with the advantages of specificity and sensitivity was used.Ten specific probes targeted at Fritillariae Cirrhosae Bulbus,F.Ussuriensis Bulbus,F.Pallidiflorae Bulbus,F.Thunbergii Bulbus and F.Hupehensis Bulbus,were designed respectively and the relative quantitative method for adulteration was established.Results:The mixture of the ten probes only amplified the specific amplification peak from these five Bulbus Fritillariae templates,indicating the probes had good specificity.The result of sensitivity test showed that the adulteration ratio of F.Cirrhosae Bulbus in one MLPA reaction was up to 10%,and the area ratio of the target amplification peaks was in accordance with the adulteration ratio.Then the adulteration test applicability of F.Cirrhosae Bulbus using MLPA method was verified by testing the samples from the market,and the adulteration ratio of the market-collected F.Cirrhosae Bulbus samples was also reflected.Conclusion:The MLPA method described in this article can effectively detect the adulteration in F.Cirrhosae Bulbus,which provids a technical support for inspection and supervision of this important traditional Chinese medicine.
引文
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[5]CHAN SW,LI SL,LIN G,et al.Pharmacokinetic study and determination of imperialine,the major bioactive component in antitussive Fritillaria cirrhosa,in rat by high-performance liquid chromatography coupled with evaporative light-scattering detector[J].Anal Biochem,2000,285(1):172
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[9]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):1830ZHANG WJ,LIU W,WEI F,et al.Study on identifying adulterants in Fritillariae Cirrhosae Bulbus with polymerase chain reactionrestriction fragment length polymorphisms(PCR-RFLP)method[J].Chin J Pharm Anal,2014,34(10):1830
[10]SCHOUTEN JP,MCELGUNN CJ,WAAIJER R,et al.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J].Nucl Acid Res,2002,30(12):e57
[11]REIJANS M,DINGEMANS G,KLAASSEN CH,et al.RespiFinder:a new multiparameter test to differentially identify fifteen respiratory viruses[J].J Clin Microbiol,2008,46(4):1232
[12]史喜菊,马贵平,乔彩霞,等.多重连接探针扩增(MLPA)技术同时检测五种病毒的研究[J].农业生物技术学报,2013,21(6):745SHI XJ,MA GP,QIAO CX,et al.Simultaneous detection of five virues by multiplex ligation-dependent probe amplification(MLPA)technology[J].J Agric Biotechnol,2013,21(6):745
[13]姚凤霞,睢瑞芳,张为民,等.多重连接探针扩增技术检测常见线粒体疾病[J].协和医学杂志2013,4(2):104YAO FX,SUI RF,ZHANG WM,et al.Multiplex ligation-dependent probe amplification technology for detection of mitochondriopathy[J].Med J Peking Union Med Coll Hosp,2013,4(2):104
[14]杨祖铭,王三南,刘敏娟,等.多重连接依赖的探针扩增技术在检测先天性心脏病遗传学病因中的应用[J].中国优生与遗传杂志,2017,6(25):20YANG ZM,WANG SN,LIU MJ,et al.Detection of major genetic causation of congenital heart disease by multiplex ligation-dependent probe amplification[J].Chin J Birth Health Hered,2017,25(6):20
[15]张丹妍,冯雪菲,戴礼猛,等.应用多重连接依赖探针扩增和微阵列比较基因组杂交术检测不明原因智力障碍患儿[J].中国优生与遗传杂志,2017,2(25):11ZHANG DY,FENG XF,DAI LM,et al.Application of MLPA and aCGHtechnology in the detection of unknown causes of mental retardation in child[J].Chin J Birth Health Hered,2017,25(2):11
[16]GUERTLER P,GOERLICH O,BUSCH U.Development of a multiplex ligation dependent probe amplification(MLPA)module for simultaneous detection of five genetically modified rapeseed events[J].Agric Sci,2014,5(6):530
[17]SAMELAK-CZAJKA A,MARSZALEK-ZENCZAK M,MARCINKOWSKA-SWOJAK M,et al.MLPA-based Analysis of copy number variation in plant populations[J].Front Plant Sci,2017,8:22
[2]刘辉,陈士林,姚辉,等.川贝母的资源学研究进展[J].中国中药杂志,2008,33(14):1645LIU H,CHEN SL,YAO H,et al.Research progress on resources in Bulbus Fritillariae Cirrhosae[J].China J Chin Mater Med,2008,33(14):1645
[3]张惠源,张志英,岳俊三.中国中药资源志要[M].北京:科学出版社,1994ZHANG HY,ZHANG ZY,YUE JS.China’s Ambition to Traditional Chinese Medicine Resources[M].Beijing:Science Press,1994
[4]于晓琳,季晖,王长礼,等.贝母的药理作用研究概况[J].中草药,2000,31(4):313YU XL,JI H,WANG CL,et al.Survey of pharmacology researches of Bulbus Fritillariae[J].Chin Tradit Herb Drugs,2000,31(4):313
[5]CHAN SW,LI SL,LIN G,et al.Pharmacokinetic study and determination of imperialine,the major bioactive component in antitussive Fritillaria cirrhosa,in rat by high-performance liquid chromatography coupled with evaporative light-scattering detector[J].Anal Biochem,2000,285(1):172
[6]桂镜生,杨树德.川贝母与平贝母的资源状况调查及市场供求分析[J].云南中医学院学报,2008,31(6):36GUI JS,YANG SD.An analysis of Bulbus Fritillariae cirrhosae and Bulbus Fritillaria ussuriensis resources survey and market supply and demand[J].J Yunnan Univ Tradit Chin Med,2008,31(6):36
[7]WANG C,LI P,DING J,et al.Simultaneous identification of Bulbus Fritillariae Cirrhosae using PCR-RFLP analysis[J].Phytomedicine,2007,14:628
[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):226XU CL,LI HJ,LI P,et al.Molecular method for the identification of Bulbus Fritillariae Cirrhosae[J].J China Pharm Univ,2010,41(3):226
[9]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):1830ZHANG WJ,LIU W,WEI F,et al.Study on identifying adulterants in Fritillariae Cirrhosae Bulbus with polymerase chain reactionrestriction fragment length polymorphisms(PCR-RFLP)method[J].Chin J Pharm Anal,2014,34(10):1830
[10]SCHOUTEN JP,MCELGUNN CJ,WAAIJER R,et al.Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification[J].Nucl Acid Res,2002,30(12):e57
[11]REIJANS M,DINGEMANS G,KLAASSEN CH,et al.RespiFinder:a new multiparameter test to differentially identify fifteen respiratory viruses[J].J Clin Microbiol,2008,46(4):1232
[12]史喜菊,马贵平,乔彩霞,等.多重连接探针扩增(MLPA)技术同时检测五种病毒的研究[J].农业生物技术学报,2013,21(6):745SHI XJ,MA GP,QIAO CX,et al.Simultaneous detection of five virues by multiplex ligation-dependent probe amplification(MLPA)technology[J].J Agric Biotechnol,2013,21(6):745
[13]姚凤霞,睢瑞芳,张为民,等.多重连接探针扩增技术检测常见线粒体疾病[J].协和医学杂志2013,4(2):104YAO FX,SUI RF,ZHANG WM,et al.Multiplex ligation-dependent probe amplification technology for detection of mitochondriopathy[J].Med J Peking Union Med Coll Hosp,2013,4(2):104
[14]杨祖铭,王三南,刘敏娟,等.多重连接依赖的探针扩增技术在检测先天性心脏病遗传学病因中的应用[J].中国优生与遗传杂志,2017,6(25):20YANG ZM,WANG SN,LIU MJ,et al.Detection of major genetic causation of congenital heart disease by multiplex ligation-dependent probe amplification[J].Chin J Birth Health Hered,2017,25(6):20
[15]张丹妍,冯雪菲,戴礼猛,等.应用多重连接依赖探针扩增和微阵列比较基因组杂交术检测不明原因智力障碍患儿[J].中国优生与遗传杂志,2017,2(25):11ZHANG DY,FENG XF,DAI LM,et al.Application of MLPA and aCGHtechnology in the detection of unknown causes of mental retardation in child[J].Chin J Birth Health Hered,2017,25(2):11
[16]GUERTLER P,GOERLICH O,BUSCH U.Development of a multiplex ligation dependent probe amplification(MLPA)module for simultaneous detection of five genetically modified rapeseed events[J].Agric Sci,2014,5(6):530
[17]SAMELAK-CZAJKA A,MARSZALEK-ZENCZAK M,MARCINKOWSKA-SWOJAK M,et al.MLPA-based Analysis of copy number variation in plant populations[J].Front Plant Sci,2017,8:22