转基因产品检测标准物质研究
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摘要
转基因生物检测标准物质是在转基因产品检测中用以校准测量装置、评价测量方法或给检测样品赋值的一种物质，其特性值足够均匀且稳定。转基因生物标准物质在转基因生物安全监管、转基因产品定性与定量检测、检测方法研究与标准化过程中是不可缺少的物质基础，可提高转基因产品检测结果可比性、有效性和溯源性。由于转基因检测标准物质的研制过程繁琐、技术要求高，研制单位较少，国际上的转基因检测有证标准物质基本上由欧盟联合研究中心下属的标准物质与测量研究所和美国的油脂化学家学会研制。我国转基因生物标准物质的研制目前尚属起步阶段，在实际检测工作中，由于转基因检测标准物质的缺乏，已经严重影响了转基因检测方法的标准化和现有标准的执行。因此，开展转基因检测标准物质的研究，可促进我国转基因检测技术和方法的发展，为我国转基因检测规章制度的建设提供技术支持，为提高我国转基因产品标准物质研制水平与国际接轨奠定基础。
另外，随着转基因技术的不断发展，转基因作物的品种、性状呈多样化趋势发展。若对盲样直接用事件特异性方法进行PCR检测，在检测成本和工作量上都不可取，而容易对未获批准的转基因作物漏检。以转基因PCR筛查作为转基因检测中的第一步，有针对性地选取部分遗传元件，对盲样进行筛查检测。筛查得到的结果，还可为下一步转化事件的鉴定提供科学依据，大大提高检测效率。
本论文针对以上转基因作物检测研究现状和存在问题，设置了以下研究内容：
1）以转基因油菜OXY235、Topas、T45，转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，对转基因作物检测基体标准物质和质粒标准物质进行研究。
2）收集国际上已经商业化应用的转基因油菜、玉米、大豆、水稻遗传转化元件信息，构建转基因作物的筛查检测矩阵，并在此基础上完成筛查策略分析。
在研究过程中取得了以下创新性成果：
1、建立和完善了转基因检测基体标准物质研制技术
在转基因检测基体标准物质研究过程中，首次在国内解决了原材料鉴定、加工、均匀性与稳定性测定与评价、量值确定与不确定度合成等关键技术问题。在原材料鉴定基础上，对原材料采用40℃恒温干燥，保证含水量小于10%。利用冷冻研磨技术保证80%以上的颗粒小于200uM。利用烘干法测定含水量，根据重量和含水量配制0%，0.5%，1%，5%四种转基因浓度。采用自动装置进行装瓶和充氮。利用荧光定量PCR检测转基因含量，并对瓶间和瓶内转基因含量进行方差分析，从而评价均匀性并合成均匀性不确定度。将材料于4℃，25℃和37℃放置1、2、4周，检验其短期稳定性，于4℃，-20℃放置1、2、4、6个月，检验其长稳定性并计算稳定性不确定，确定材料可于常温运输，可于4℃稳定储存半年以上。最后通过8家实验室联合定值的策略，对材料的转基因含量进行检测，材料的不确定度则来自于均匀性、稳定性和定值，通过公式合成了本批标准物质的不确定度，并完成了赋值全过程。
2、制备完成3种转化体的基体标准物质，每种转化体4个浓度
以转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，每个转化体获得0%，0.5%，1%，5%四种转基因浓度的基体标准物质，每个浓度100瓶。经过量值测定和不确定度分析，标准物质具有所要求的均匀性、稳定性，并确定了量值和不确定度，可用于转基因成分定性和定量检测。
3、建立和完善了转基因检测质粒标准物质制备技术
通过研究，解决了载体的设计与构建、适合性测试、可替代性等关键问题。在构建质粒载体前，根据转基因植物的旁侧序列和内标准基因，结合国内外发布的标准检测方法，确定检测的靶标片段。通过PCR、酶切、连接等分子生物学方法将旁侧序列、内标准基因、以及将这两个片段隔开的间隔序列克隆至骨架载体pBluescript II SK (+)中，最后对构建完毕的6个质粒载体进行PCR、酶切和测序确认，并以测序所得结果对质粒中外源和内标片段比例赋值。此外，以质粒为模板，利用定性和定量PCR引物检测质粒扩增的特异性、灵敏度，证明了质粒的PCR扩增适合性。分别用质粒和基因组DNA绘制标准曲线，比较了质粒和基因组标准曲线的差异。以构建的标准曲线对盲样中转基因含量进行检测，并计算校正系数和校正方程，比较不同方法的准确度和精确度，确定了质粒在定量检测中的应用方法，为质粒的可替代性问题提供了一种可行的解决方案。
4、制备了6种质粒标准分子
以转基因油菜OXY235、Topas、T45，转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，制备了6个不同的质粒标准分子，每种质粒标准分子制备了1000管，以107copies/μL的浓度每管500μL的量分装。经实验证明这种质粒标准分子具有良好的均匀性、稳定性、PCR扩增性，是一种良好的转基因检测标准物质。
5、建立了商业化转化体的基因元件矩阵表，完成了转基因筛查策略研究
根据加拿大转基因数据库、欧洲转基因生物指南、上海市转基因生物安全检测评估共享服务平台上公布的所有转基因油菜、玉米、大豆、水稻外源基因的分子特征，对转入的外源目的基因、启动子、终止子、筛选标记基因进行统计，建立遗传转化元件的矩阵表。经分析，对转基因油菜利用P-FMV35S、P-CaMV35S、BAR三个元件；对转基因玉米利用P-CaMV35S和T-NOS；对转基因大豆利用P-CaMV35S，Cry1Ac，gat，T-35S，T-E9；对转基因水稻利用P-CaMV35S，T-NOS，筛查率可达100%。本研究针对各物种建立了转基因筛查检测和快速定位策略，为转基因作物的筛查检测提供了科学的依据和重要的参考。
Reference material (RM) for detection of Genetically modified organisms (GMO) is a material orsubstance one or more of whose property values are sufficiently homogeneous and well established tobe used in GMO detection for the calibration of an apparatus, the assessment of a measurement method,or for assigning values to materials. GMO detection RM is indispensable to GMO safety control, GMOqualitative and quantitative detection, and standardization of GMO detection method. It can be used toimprove the comparability, validity and traceability of GMO detection. Procedure of GMO RMpreparation is cumbersome and technically demanding. By now, Only a few departments havedeveloped GMO CRM, and most of the GMO CRM have been developed by EU Joint Research Centre(Institute for Reference Materials and Measurements，IRMM) and American Oil Chemists' Society(AOCS). In China, research and development of GMO detection RM is still in infancy. Lack of RM inroutine work has hindered standardization of GMO detection technology and the implementation ofexisting standards. Therefore, studies on GMO detection RM will promote the development oftechniques and methods for GMO detection, provide technical support for the construction of theregulations of GMO detection, and lay a foundation for developing China's GMO detection RM,towards a international level.
In addition, with the development of transgenic technology, varieties and traits of GM plant weremore and more diverse. If we directly detect the blind sample with event-specific method, the testingcost and effort are unacceptable; also it is possible to miss out un-approved GMO. Using screeningdetection as the first step and optimizing the combination of the detection targets with certain geneticelements can greatly improve the detection efficiency. Screening results can also provide a scientificreference for event-specific detection.
On the status quo of GMO detection, the dissertation here is composed of two main parts. One is todevelop matrix RM and plasmid RM for detection of GM rapeseed OXY235, Topas, T45, GM soybeanMON89788, GM maize MIR604, T25. And the other is to collect the information of inserted geneelements for the internationally commercialized events of transgenic rapeseed, maize, soybeans and riceto establish molecular characteristics matrix of these transgenic events.
The major research contents and the points of innovation in the study are as follows:
1. Development and improvement of technology for production of GMO matrix RM
Through the study of GMO detection matrix RM, we have solved the key technical issues in rawmaterial identification, processing, evaluation of homogeneity and stability, determination of value andcalculation of uncertainty. Based on identification of raw materials, the seed were dried under constanttemperature at40℃, and kept water content below10%. The seeds were milled using a cryo-grindingvibrating mill to ensure that more than80%of particles less than200uM. Four transgenicconcentrations were prepared according to the weight and water content of GM and Non-GM material.Bottling was implemented by automatic devices. Random samples of the prepared RM were taken, and GM content was analyzed using real-time PCR. Homogeneity and related uncertainties were calculated,indicating that these RMs were homogeneous. Short-term stability was studied at4°C,25°C and37°Cfor1,2,4weeks, and long-term stability at4°C and-20°C for1,2,4,6months. The results indicatedthat these RMs can be shipped under ambient conditions and stored at4°C or-20°C for6months ormore. At last, the GM content was determined by inter-laboratory study, and the expanded uncertaintyof certified value was comprised with homogeneity, long-term stability and inter-laboratory studystandard uncertainty. A complete set of the techniques for preparation of matrix RMs have beenesdablished.
2. Preparation of matrix RM for three transgenic events, each event with4concentrations
The matrix RMs was prepared for GM events of soybean MON89788, maize MIR604and T25.For each event, four concentrations (0%,0.5%,1%, and5%), and100bottles for each concentrationwere prepared. Through determination of value and uncertainty, these RMs were proved to besufficiently homogenous and stable, and can be used in GMO qualitative and quantitative detection.
3. Development and improvement of technology for production of GMO plasmid RM
A strategy to construct plasmid molecule, and to test the suitability and commutability of theconstructed plasmid in PCR assays has been established. In plasmid vector construction, firstly, theflanking sequence, endogenous reference gene and interval sequence were determined according toexisting standard detection methods. Secondly, these three fragments were clone into pBluescript II SK(+) vector through molecular biology methods, such as PCR, restriction enzyme digestion and ligation.Finally, the vector was confirmed by PCR, enzyme digestion and sequencing. The certified value ofplasmid RM was the number of cloned DNA fragment for the flanking sequence and endogenousreference gene targets per plasmid. In addition, the specificity and sensitivity of plasmid template inqualitative PCR assays was test, similar results were obtained when using pDNA and gDNA as template.In order to study the commutability of pDNA in quantitative PCR, standard curves of pDNA and gDNAwere constructed and compared; both of these standard curves were used to calculate conversion factorand conversion equation, and to measure GM content of blind samples. We put forward a new andfeasible solution for commutability of pDNA in this study.
4. Preparation of plasmid RMs for six transgenic events
Six different plasmid reference molecules were constructed for GM rapeseed OXY235, Topas, T45,soybean MON89788, maize MIR604and T25. The plasmid DNA was diluted to a concentration107copies/μL, and filled500μL in to a bottle,1000bottles of plasmid RM were prepared for each plasmid.The experiment results proved that the plasmid RM have a good homogenous and stability, and can bewell amplified in PCR assays. So plasmid DNA is good as a RM for GMO detection.
5. Construction of the inserted gene elements matrix table of commercialized transgenic events,and establishment of the screening strategy for GMO detection
Matrix tables were constructed on the basis of target gene, promoter, terminator and select markergene by retrieving Canadian GM crop database, European GMO compass and GMO Detection methodDatabase (GMDD), domestic and foreign commercialized events of transgenic rapeseed, maize, soybean and rice were collected. Based on the frequency of application of these gene elements,P-FMV35S, P-CaMV35S and BAR were used in rapeseed screening detection. The combinations ofscreening detection targets were optimized to be P-CaMV35S and T-NOS for maize, P-CaMV35S,Cry1Ac, gat, T-35S and T-E9for soybean, P-CaMV35S and T-NOS for rice. Using these elements, theGMO can be reliably detected once. This study has established a strategy for transgenic screeningdetection and for rapid identification of GM events, providing a scientific basis and an importantreference for the screening detection of GM crops.
另外，随着转基因技术的不断发展，转基因作物的品种、性状呈多样化趋势发展。若对盲样直接用事件特异性方法进行PCR检测，在检测成本和工作量上都不可取，而容易对未获批准的转基因作物漏检。以转基因PCR筛查作为转基因检测中的第一步，有针对性地选取部分遗传元件，对盲样进行筛查检测。筛查得到的结果，还可为下一步转化事件的鉴定提供科学依据，大大提高检测效率。
本论文针对以上转基因作物检测研究现状和存在问题，设置了以下研究内容：
1）以转基因油菜OXY235、Topas、T45，转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，对转基因作物检测基体标准物质和质粒标准物质进行研究。
2）收集国际上已经商业化应用的转基因油菜、玉米、大豆、水稻遗传转化元件信息，构建转基因作物的筛查检测矩阵，并在此基础上完成筛查策略分析。
在研究过程中取得了以下创新性成果：
1、建立和完善了转基因检测基体标准物质研制技术
在转基因检测基体标准物质研究过程中，首次在国内解决了原材料鉴定、加工、均匀性与稳定性测定与评价、量值确定与不确定度合成等关键技术问题。在原材料鉴定基础上，对原材料采用40℃恒温干燥，保证含水量小于10%。利用冷冻研磨技术保证80%以上的颗粒小于200uM。利用烘干法测定含水量，根据重量和含水量配制0%，0.5%，1%，5%四种转基因浓度。采用自动装置进行装瓶和充氮。利用荧光定量PCR检测转基因含量，并对瓶间和瓶内转基因含量进行方差分析，从而评价均匀性并合成均匀性不确定度。将材料于4℃，25℃和37℃放置1、2、4周，检验其短期稳定性，于4℃，-20℃放置1、2、4、6个月，检验其长稳定性并计算稳定性不确定，确定材料可于常温运输，可于4℃稳定储存半年以上。最后通过8家实验室联合定值的策略，对材料的转基因含量进行检测，材料的不确定度则来自于均匀性、稳定性和定值，通过公式合成了本批标准物质的不确定度，并完成了赋值全过程。
2、制备完成3种转化体的基体标准物质，每种转化体4个浓度
以转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，每个转化体获得0%，0.5%，1%，5%四种转基因浓度的基体标准物质，每个浓度100瓶。经过量值测定和不确定度分析，标准物质具有所要求的均匀性、稳定性，并确定了量值和不确定度，可用于转基因成分定性和定量检测。
3、建立和完善了转基因检测质粒标准物质制备技术
通过研究，解决了载体的设计与构建、适合性测试、可替代性等关键问题。在构建质粒载体前，根据转基因植物的旁侧序列和内标准基因，结合国内外发布的标准检测方法，确定检测的靶标片段。通过PCR、酶切、连接等分子生物学方法将旁侧序列、内标准基因、以及将这两个片段隔开的间隔序列克隆至骨架载体pBluescript II SK (+)中，最后对构建完毕的6个质粒载体进行PCR、酶切和测序确认，并以测序所得结果对质粒中外源和内标片段比例赋值。此外，以质粒为模板，利用定性和定量PCR引物检测质粒扩增的特异性、灵敏度，证明了质粒的PCR扩增适合性。分别用质粒和基因组DNA绘制标准曲线，比较了质粒和基因组标准曲线的差异。以构建的标准曲线对盲样中转基因含量进行检测，并计算校正系数和校正方程，比较不同方法的准确度和精确度，确定了质粒在定量检测中的应用方法，为质粒的可替代性问题提供了一种可行的解决方案。
4、制备了6种质粒标准分子
以转基因油菜OXY235、Topas、T45，转基因大豆MON89788，转基因玉米MIR604、T25为研究对象，制备了6个不同的质粒标准分子，每种质粒标准分子制备了1000管，以107copies/μL的浓度每管500μL的量分装。经实验证明这种质粒标准分子具有良好的均匀性、稳定性、PCR扩增性，是一种良好的转基因检测标准物质。
5、建立了商业化转化体的基因元件矩阵表，完成了转基因筛查策略研究
根据加拿大转基因数据库、欧洲转基因生物指南、上海市转基因生物安全检测评估共享服务平台上公布的所有转基因油菜、玉米、大豆、水稻外源基因的分子特征，对转入的外源目的基因、启动子、终止子、筛选标记基因进行统计，建立遗传转化元件的矩阵表。经分析，对转基因油菜利用P-FMV35S、P-CaMV35S、BAR三个元件；对转基因玉米利用P-CaMV35S和T-NOS；对转基因大豆利用P-CaMV35S，Cry1Ac，gat，T-35S，T-E9；对转基因水稻利用P-CaMV35S，T-NOS，筛查率可达100%。本研究针对各物种建立了转基因筛查检测和快速定位策略，为转基因作物的筛查检测提供了科学的依据和重要的参考。
Reference material (RM) for detection of Genetically modified organisms (GMO) is a material orsubstance one or more of whose property values are sufficiently homogeneous and well established tobe used in GMO detection for the calibration of an apparatus, the assessment of a measurement method,or for assigning values to materials. GMO detection RM is indispensable to GMO safety control, GMOqualitative and quantitative detection, and standardization of GMO detection method. It can be used toimprove the comparability, validity and traceability of GMO detection. Procedure of GMO RMpreparation is cumbersome and technically demanding. By now, Only a few departments havedeveloped GMO CRM, and most of the GMO CRM have been developed by EU Joint Research Centre(Institute for Reference Materials and Measurements，IRMM) and American Oil Chemists' Society(AOCS). In China, research and development of GMO detection RM is still in infancy. Lack of RM inroutine work has hindered standardization of GMO detection technology and the implementation ofexisting standards. Therefore, studies on GMO detection RM will promote the development oftechniques and methods for GMO detection, provide technical support for the construction of theregulations of GMO detection, and lay a foundation for developing China's GMO detection RM,towards a international level.
In addition, with the development of transgenic technology, varieties and traits of GM plant weremore and more diverse. If we directly detect the blind sample with event-specific method, the testingcost and effort are unacceptable; also it is possible to miss out un-approved GMO. Using screeningdetection as the first step and optimizing the combination of the detection targets with certain geneticelements can greatly improve the detection efficiency. Screening results can also provide a scientificreference for event-specific detection.
On the status quo of GMO detection, the dissertation here is composed of two main parts. One is todevelop matrix RM and plasmid RM for detection of GM rapeseed OXY235, Topas, T45, GM soybeanMON89788, GM maize MIR604, T25. And the other is to collect the information of inserted geneelements for the internationally commercialized events of transgenic rapeseed, maize, soybeans and riceto establish molecular characteristics matrix of these transgenic events.
The major research contents and the points of innovation in the study are as follows:
1. Development and improvement of technology for production of GMO matrix RM
Through the study of GMO detection matrix RM, we have solved the key technical issues in rawmaterial identification, processing, evaluation of homogeneity and stability, determination of value andcalculation of uncertainty. Based on identification of raw materials, the seed were dried under constanttemperature at40℃, and kept water content below10%. The seeds were milled using a cryo-grindingvibrating mill to ensure that more than80%of particles less than200uM. Four transgenicconcentrations were prepared according to the weight and water content of GM and Non-GM material.Bottling was implemented by automatic devices. Random samples of the prepared RM were taken, and GM content was analyzed using real-time PCR. Homogeneity and related uncertainties were calculated,indicating that these RMs were homogeneous. Short-term stability was studied at4°C,25°C and37°Cfor1,2,4weeks, and long-term stability at4°C and-20°C for1,2,4,6months. The results indicatedthat these RMs can be shipped under ambient conditions and stored at4°C or-20°C for6months ormore. At last, the GM content was determined by inter-laboratory study, and the expanded uncertaintyof certified value was comprised with homogeneity, long-term stability and inter-laboratory studystandard uncertainty. A complete set of the techniques for preparation of matrix RMs have beenesdablished.
2. Preparation of matrix RM for three transgenic events, each event with4concentrations
The matrix RMs was prepared for GM events of soybean MON89788, maize MIR604and T25.For each event, four concentrations (0%,0.5%,1%, and5%), and100bottles for each concentrationwere prepared. Through determination of value and uncertainty, these RMs were proved to besufficiently homogenous and stable, and can be used in GMO qualitative and quantitative detection.
3. Development and improvement of technology for production of GMO plasmid RM
A strategy to construct plasmid molecule, and to test the suitability and commutability of theconstructed plasmid in PCR assays has been established. In plasmid vector construction, firstly, theflanking sequence, endogenous reference gene and interval sequence were determined according toexisting standard detection methods. Secondly, these three fragments were clone into pBluescript II SK(+) vector through molecular biology methods, such as PCR, restriction enzyme digestion and ligation.Finally, the vector was confirmed by PCR, enzyme digestion and sequencing. The certified value ofplasmid RM was the number of cloned DNA fragment for the flanking sequence and endogenousreference gene targets per plasmid. In addition, the specificity and sensitivity of plasmid template inqualitative PCR assays was test, similar results were obtained when using pDNA and gDNA as template.In order to study the commutability of pDNA in quantitative PCR, standard curves of pDNA and gDNAwere constructed and compared; both of these standard curves were used to calculate conversion factorand conversion equation, and to measure GM content of blind samples. We put forward a new andfeasible solution for commutability of pDNA in this study.
4. Preparation of plasmid RMs for six transgenic events
Six different plasmid reference molecules were constructed for GM rapeseed OXY235, Topas, T45,soybean MON89788, maize MIR604and T25. The plasmid DNA was diluted to a concentration107copies/μL, and filled500μL in to a bottle,1000bottles of plasmid RM were prepared for each plasmid.The experiment results proved that the plasmid RM have a good homogenous and stability, and can bewell amplified in PCR assays. So plasmid DNA is good as a RM for GMO detection.
5. Construction of the inserted gene elements matrix table of commercialized transgenic events,and establishment of the screening strategy for GMO detection
Matrix tables were constructed on the basis of target gene, promoter, terminator and select markergene by retrieving Canadian GM crop database, European GMO compass and GMO Detection methodDatabase (GMDD), domestic and foreign commercialized events of transgenic rapeseed, maize, soybean and rice were collected. Based on the frequency of application of these gene elements,P-FMV35S, P-CaMV35S and BAR were used in rapeseed screening detection. The combinations ofscreening detection targets were optimized to be P-CaMV35S and T-NOS for maize, P-CaMV35S,Cry1Ac, gat, T-35S and T-E9for soybean, P-CaMV35S and T-NOS for rice. Using these elements, theGMO can be reliably detected once. This study has established a strategy for transgenic screeningdetection and for rapid identification of GM events, providing a scientific basis and an importantreference for the screening detection of GM crops.
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