基于PCR技术的DNA分析测试关键要素
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摘要
大多数国家对转基因生物研究与产业化日趋积极,把发展生物技术作为支撑发展、引领未来的战略选择,力求抢占新一轮经济和科技革命的先机与制高点。随之而来的是公众对转基因产品安全性的关注。转基因生物成分分析作为安全性评价的重要内容,发挥着举足轻重的作用,而基于PCR技术的转基因产品核酸成分分析是最经典、最广泛使用的方法。虽然此方法已普遍应用,但国内外鲜有较为全面的阐述此类分析测试要素的报道。为此,本综述对国内外相关方法的文献资料进行了梳理、比对和研究,结合已有的分析测试实验室的经验,提出了PCR测试体系的关键要素主要是标准和指南、方法的确认、标准物质及能力验证等四个方面,且在文中简明扼要的论述了各个部分的要点,供转基因核酸成分分析及相关领域工作者参考。
The research and industrialization of genetically modified organism(GMO) had generally gained much attention in most countries. Most countries regarded the development of biotechnology as a strategic choice to support development and led the future, and strived to seize the opportunities and commanding heights of a new round of economic and technological revolution. However, safety issues of GMO attracted the people's attention.As a main content in safety assessment, composition analysis of GMO was indispensable. The component analysis of nucleic acid in transgenic product based on polymerase chain reaction(PCR) was a principal approach and widely used in this area. Although this method has been widely used, there were few comprehensive reports to explain such analytical testing element at home and abroad. For this purpose, this review has teased, compared and studied the literature of relevant methods at home and abroad. Based on the analysis experience of our lab, we came up with the key elements of the PCR test included standards and guidelines, methods validation, standard material and capacity verification. In addition, the main points of the above four parts were discussed, which could be a reference for GMO analysis and provide reference for workers in related fields.
The research and industrialization of genetically modified organism(GMO) had generally gained much attention in most countries. Most countries regarded the development of biotechnology as a strategic choice to support development and led the future, and strived to seize the opportunities and commanding heights of a new round of economic and technological revolution. However, safety issues of GMO attracted the people's attention.As a main content in safety assessment, composition analysis of GMO was indispensable. The component analysis of nucleic acid in transgenic product based on polymerase chain reaction(PCR) was a principal approach and widely used in this area. Although this method has been widely used, there were few comprehensive reports to explain such analytical testing element at home and abroad. For this purpose, this review has teased, compared and studied the literature of relevant methods at home and abroad. Based on the analysis experience of our lab, we came up with the key elements of the PCR test included standards and guidelines, methods validation, standard material and capacity verification. In addition, the main points of the above four parts were discussed, which could be a reference for GMO analysis and provide reference for workers in related fields.
引文
Broeders S.,Huber I.,Grohmann L.,Berben G.,Taverniers I.,Mazzara M.,and Moriset D.,2014,Guidelines for validation of qualitative real-time PCR methods,Trends in Food Science and Technology,37(2):115-126
Burns M.,Corbisier P.,Wiseman G.,Valdivia H.,McDonald P.,Bowler P.,Ohara K.,Schimmel H.,Charels D.,Damant A.,and Harris N.,2006,Comparison of plasmid and genomic DNA calibrants for the quantification of genetically modified ingredients,Eur.Food Res.Technol.,224(1):249-258
Cai J.,Li H.,Hu M.L.,Fu Yang.,Wang L.,and Wang L.,2016,Research progress in techniques for the detection and analysis of genetically modified ingredients,Shiping Anquan Zhiliang Jiance Xuebao(Journal of Food Saety and Quality)7(2):706-714(蔡军,李慧,胡梦龙,傅洋,汪磊,王璐,2016,转基因成分分析检测技术研究进展,食品安全质量检测学报,7(2):706-714)
Caprioara-Buda M.,Meyer W.,Jeynov B.,Corbisier P.,Trapmann S.,and Emons H.,2012,Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms,Analytical and Bioanalytical Chemistry,,404(1):29-42
Dong L.H.,Sui Z.W.,Yu X.B.,and Li L.,2012,Characterization of genetically modified maize line NK603 matrix reference material,Jiliang Xuebao(Acta Metrologica Sinica),33(1):1-6(董莲华,隋志伟,余效波,李亮,王晶,2012,转基因玉米NK603基体标准物质协同定值研究,计量学报,33(1):1-6)
Du Z.X.,Jiao Y.,Zhang L.L.,Zhu P.Y.,and Fu W.,2017,Development of quantitative detection techniques of genetically modified organisms,Shiping Gongye Keji(Science and Technology of Food Industry),38(10):379-384(杜智欣,焦悦,张亮亮,朱鹏宇,付伟,2017,转基因成分定量检测技术研究进展,食品工业科技,38(10):379-384)
International Standard(ISO)17025,2017,General requirements for the competence of testing and calibration laboratories,International Organisation for Standardisation,5(15):1-27
International standard(ISO)21569,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methods,International Organisation for Standardisation,6(15):1-67
International standard(ISO)21570,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Quantitative nucleic acid based methods,International Organisation for Standardisation,12(15):1-100
International standard(ISO)21571,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Nucleic acid extraction,International Organisation for Standardisation,2(15):1-41
International standard(ISO)24276,2006,Foodstuffs.Methods of analysis for the detection of genetically modified organisms and derived products-General requirements,International Organisation for Standardisation,2(1):1-16
International Standard(ISO)5725,1994,Accuracy(trueness and precision)of measurement methods and results,International Organization for Standardization,12(15):1-42
Li L.,Wang J.,Zang C.,Sui Z.W.,and Zhao Z.Y.,2013,Development of a reference plasmid for the quantitative detection of genetically modified rice Huahui 1,Shengwu Jishu Tongbao(Biotechnology Bulletin),(1):96-101(李亮,王晶,臧超,隋志伟,赵正宜,2013,转基因水稻华恢1号检测用质粒标准分子研制,生物技术通报,(1):96-101)
Li L.,Zang C.,Wang J.,Sui Z.W.,Zhao Z.Y.,and Dong L.H.,2012,Construction of a reference plasmid for the quantification of genetically modified rice kemingdao 2 and uncertainty evaluation,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),32(10):19-24(李亮,臧超,王晶,隋志伟,赵正宜,董莲华,2012,转基因水稻克螟稻2号定量检测用质粒标准分子研制及不确定度评价,中国生物工程杂志,32(10):19-24)
Lubeck M.,and Haugaard H.,eds.,2002,Detection of genetically modified plants,The Danish Forest and Nature,Agency Press,Copenhagen,Denmark,pp.1-30
Meng Y.,Lu X.,Shu L.,Wang S.,Zhang D.,and Yang L.,2012,Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize,an interlaboratory study,Agric.Food Chem.,60(1):23-28
譕el J.,Milavec M.,Morisset D.,Plan D.,Van den Eede G.,and Gruden K.,2012,How to reliably test for GMOs,Spring Briefes in Food Health and Nutrition,(1):1-95
Burns M.,Corbisier P.,Wiseman G.,Valdivia H.,McDonald P.,Bowler P.,Ohara K.,Schimmel H.,Charels D.,Damant A.,and Harris N.,2006,Comparison of plasmid and genomic DNA calibrants for the quantification of genetically modified ingredients,Eur.Food Res.Technol.,224(1):249-258
Cai J.,Li H.,Hu M.L.,Fu Yang.,Wang L.,and Wang L.,2016,Research progress in techniques for the detection and analysis of genetically modified ingredients,Shiping Anquan Zhiliang Jiance Xuebao(Journal of Food Saety and Quality)7(2):706-714(蔡军,李慧,胡梦龙,傅洋,汪磊,王璐,2016,转基因成分分析检测技术研究进展,食品安全质量检测学报,7(2):706-714)
Caprioara-Buda M.,Meyer W.,Jeynov B.,Corbisier P.,Trapmann S.,and Emons H.,2012,Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms,Analytical and Bioanalytical Chemistry,,404(1):29-42
Dong L.H.,Sui Z.W.,Yu X.B.,and Li L.,2012,Characterization of genetically modified maize line NK603 matrix reference material,Jiliang Xuebao(Acta Metrologica Sinica),33(1):1-6(董莲华,隋志伟,余效波,李亮,王晶,2012,转基因玉米NK603基体标准物质协同定值研究,计量学报,33(1):1-6)
Du Z.X.,Jiao Y.,Zhang L.L.,Zhu P.Y.,and Fu W.,2017,Development of quantitative detection techniques of genetically modified organisms,Shiping Gongye Keji(Science and Technology of Food Industry),38(10):379-384(杜智欣,焦悦,张亮亮,朱鹏宇,付伟,2017,转基因成分定量检测技术研究进展,食品工业科技,38(10):379-384)
International Standard(ISO)17025,2017,General requirements for the competence of testing and calibration laboratories,International Organisation for Standardisation,5(15):1-27
International standard(ISO)21569,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methods,International Organisation for Standardisation,6(15):1-67
International standard(ISO)21570,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Quantitative nucleic acid based methods,International Organisation for Standardisation,12(15):1-100
International standard(ISO)21571,2005,Methods of analysis for the detection of genetically modified organisms and derived products-Nucleic acid extraction,International Organisation for Standardisation,2(15):1-41
International standard(ISO)24276,2006,Foodstuffs.Methods of analysis for the detection of genetically modified organisms and derived products-General requirements,International Organisation for Standardisation,2(1):1-16
International Standard(ISO)5725,1994,Accuracy(trueness and precision)of measurement methods and results,International Organization for Standardization,12(15):1-42
Li L.,Wang J.,Zang C.,Sui Z.W.,and Zhao Z.Y.,2013,Development of a reference plasmid for the quantitative detection of genetically modified rice Huahui 1,Shengwu Jishu Tongbao(Biotechnology Bulletin),(1):96-101(李亮,王晶,臧超,隋志伟,赵正宜,2013,转基因水稻华恢1号检测用质粒标准分子研制,生物技术通报,(1):96-101)
Li L.,Zang C.,Wang J.,Sui Z.W.,Zhao Z.Y.,and Dong L.H.,2012,Construction of a reference plasmid for the quantification of genetically modified rice kemingdao 2 and uncertainty evaluation,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),32(10):19-24(李亮,臧超,王晶,隋志伟,赵正宜,董莲华,2012,转基因水稻克螟稻2号定量检测用质粒标准分子研制及不确定度评价,中国生物工程杂志,32(10):19-24)
Lubeck M.,and Haugaard H.,eds.,2002,Detection of genetically modified plants,The Danish Forest and Nature,Agency Press,Copenhagen,Denmark,pp.1-30
Meng Y.,Lu X.,Shu L.,Wang S.,Zhang D.,and Yang L.,2012,Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize,an interlaboratory study,Agric.Food Chem.,60(1):23-28
譕el J.,Milavec M.,Morisset D.,Plan D.,Van den Eede G.,and Gruden K.,2012,How to reliably test for GMOs,Spring Briefes in Food Health and Nutrition,(1):1-95
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