The safety supervision of genetically modified organisms (GMO) is the guarantee for the healthy development of the biotechnology, and the reference materials (RM) are the material basis for GMOs detection. The lack of RMs makes it difficult to ensure the accuracy, reliability and comparability of the test data. GM maize MIR604 is approved for import as a raw material in China. The safety supervision of GM maize MIR604 urgently requires to prepare RMs. The pure GM maize MIR604 matrix RMs were prepared through the steps of raw material identification, grinding, sieving and water content determination. The results of homogeneity test and stability test showed that the RMs in this batch had good homogeneity within the bottle and between the bottles, the property value of the RMs was stable, allowing them to be transported at room temperature for one month, as even for six months verified by the long-term stability test. The copy number ratio of the transgenic DNA to the total DNA was collaboratively characterized by nine laboratories using the duplex digital PCR of MIR604/Adh1. The certified value was 0.50. The various uncertainty components of the RM characterization results were fully evaluated, and the expanded uncertainty was combined to be 0.06 (copy/copy). The specification of RMs of this batch is 1 g bottle–1 and the minimum sample size is 100 mg. This batch of RMs can be used in the fields of safety supervision and labelling of MIR604, and laboratory quality control.

转基因检测标准物质是获得准确、可靠、可比检测结果的物质保证,检测实验室通过使用标准物质保证转基因检测结果的量值溯源.以籽粒为原材料研制纯品粉末标准物质,工艺相对简单,量值可靠,是转基因检测标准物质的一种重要类型,广泛用于转基因产品的定量检测.本研究以转基因大豆(Glycine max)MON89788纯合种子为原材料,通过原材料鉴定、研磨、粒度测定、含水量测定、均匀性初检等步骤,制备出大豆MON89788纯品基体标准物质,规格为每瓶1 g.本批标准物质粉末中小于200μm的颗粒占80％以上,粉末含水量为(4.53±0.03)％,低于规定的10％.标准物质均匀性良好,最小取样量为100 mg;可在常温下运输1个月,长期稳定性达到6个月.本批标准物质的特性量值转基因DNA与总DNA的拷贝数比值(copy/copy)由9家实验室采用数字PCR联合测定,量值为1.00,扩展不确定度为0.07.本批标准物质可用于转基因大豆MON89788的定性和定量检测,以及MON89788特异性检测方法的评价和实验室质量控制.

<span id="ChDivSummary" name="ChDivSummary" class="abstract-text">针对如何更加科学精准地检测转基因植物的问题,采用比较研究的方法,对国内外转基因植物的检测方法及其标准化进行研究。结果表明:1)转基因植物及其产品的检测,主要包括转基因植物的产品成分检测、环境安全检测、食用安全检测3个方面;2)目前国内外转基因植物检测方法均处于先进水平,但随着不断涌现的转基因植物及产品,检测技术仍需更新换代;3)转基因植物检测技术研发必须与其标准化及安全管理协调进行,从而保证转基因植物及其产品在我国长期有效的安全监管。综上,转基因植物检测技术及其标准化仍需要不断开发和创新,为国家对转基因产品的安全监管提供更有力的技术保障。</span>

Adult Chrysoperla sinica Tjeder is a common pollen feeder in maize fields. They are thus directly exposed to insecticidal proteins by consumption of genetically engineered maize pollen containing Bacillus thuringiensis（Bt） proteins. Here we assessed the potential effects of Cry1 Ab/2 Aj-or Cry1 Ac-containing Bt maize pollen on the fitness of adult C. sinica via a dietary-exposure assay under laboratory conditions. Survival, pre-oviposition, fecundity and adult dry weight did not differ between adult C. sinica consuming Bt or the corresponding non-Bt maize pollen. The stability of the Cry protein in the food sources and uptake of the Cry protein by adult C. sinica during the feeding experiment were confirmed by ELISA. These results demonstrate that adult C. sinica are not affected by the consumption of Cry1 Ab/2 Aj-or Cry1 Ac-containing maize pollen, suggesting that production of Bt maize expressing cry1 Ab/2 Aj or cry1 Ac genes will pose a negligible risk to adult C. sinica.

The common soil arthropod Folsomia candida can survive well when fed only maize pollen and thus may be exposed to insecticidal proteins by ingesting insect-resistant genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins when being released into the soil. Laboratory experiments were conducted to assess the potential effects of Cry1Ab/Cry2Aj-producing transgenic Bt maize (Shuangkang 12-5) pollen on F. candida fitness. Survival, development, and the reproduction were not significantly reduced when F. candida fed on Bt maize pollen rather than on non-Bt maize pollen, but these parameters were significantly reduced when E candida fed on non-Bt maize pollen containing the protease inhibitor E-64 at 75 mu g/g pollen. The intrinsic rate of increase (r(m)) was not significantly reduced when F. candida fed on Bt maize pollen but was significantly reduced when F. candida fed on non-Bt maize pollen containing E-64. The activities of antioxidant-related enzymes in F candida were not significantly affected when F candida fed on Bt maize pollen but were significantly increased when F. candida fed on non-Bt pollen containing E-64. The results demonstrate that consumption of Bt maize pollen containing Cry1Ab/Cry2Aj has no lethal or sublethal effects on F candida. (C) 2017 Elsevier Ltd. All rights reserved.

Genome editing technologies using sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites,and the DSBs can be repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways with the help of artificially engineered nucleases,which can be employed to achieve targeted genome modifications such as gene mutation,gene insertion,gene replacement or chromosome rearrangement.There are three major artificially engineered nucleases including zinc finger nuclease (ZFN),transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) systems.The genome editing techniques have been widely used in the fields of plant gene function research,molecular breeding,and etc.The genome editing crops (GEC) show promising application prospects compared with traditional genetically modified organism (GMO).The GECs with good traits have been gradually transferred from labs to fields.Herein,the principles,characters,and applications of these three mainly used techniques in animal and plants genome editing have been described.The advantages and disadvantages compared with conventional transgenic technique were also discussed,including the safety came from the off-target effects.The management regulations of GECs of different countries in global area were elaborated.Finally,the safety management of GECs and their products in China were discussed combined with Chinese current regulations on the safety management of GMOs,so as to facilitate the development and commercialization of GECs and their products.

21世纪是生物技术的世纪,加快转基因技术研发已成为世界各国增强核心竞争力的战略决策.介绍了全球转基因作物的研发、种植及应用状况.农业生物技术产业进入全球化布局的新阶段,各国纷纷规划和布局,以期在渐趋垄断的技术与市场中争得先机.经过20年的发展,我国在生物技术研发领域已取得长足进步,抗虫棉大面积推广,产生了良好的经济效益和社会效益.但受消极舆论环境等因素的影响,推动其他转基因作物产业化的进程缓慢.我国是一个人口大国,粮食供需矛盾突出,大力发展生物技术产业是我国未来的必然选择.我国一方面要在确保安全的基础上坚持走自主创新之路;另一方面政府要建立及时的信息披漏机制,营造良好的舆论环境,从国计民生出发,在科学的基础上自主决策,推动我国生物技术产业的健康、有序发展.

转基因技术是国家战略,推动转基因产业化应同时注重科学研究、推广应用、安全管理和科学普及.文章介绍了我国转基因生物安全管理相关的法律法规、立法背景和现行转基因生物安全评价与管理体系,并通过对我国重大转基因研发及管理政策的分析,提出了我国转基因安全管理新动态.

转基因产品成分检测是转基因生物安全管理和标识的重要支撑,而转基因生物标准物质是获得准确、可靠、具有可比性检测结果的保证。转基因大豆（Glycine max）MON89788为我国批准进口用作加工原料的农业转基因生物,因此,制备转基因大豆MON89788基体标准物质是对其进行安全管理所必需的。本研究通过将转基因大豆MON89788和其受体材料A3244分别冷冻研磨成粉末,按质量分数称量配制,得到转基因含量为49.3 g/kg的标准物质。采用方差分析法（F检验法）进行均匀性检验,统计量F值为1.618,小于临界值F （0.05,11,24） ,标准物质均匀性良好;短期稳定性考察结果表明,标准物质可在常温下稳定储存4周,在-20℃下长期稳定性达到30个月。本批MON89788基体标准物质有两个特性量值,一是基于重量法配比混合的粉末质量分数,量值为49.3 g/kg,扩展不确定度为2.0 g/kg;二是基于q RT-PCR 8家实验室联合定值的转基因DNA与总DNA的质量分数,量值为49.8 ng/μg,扩展不确定度为5.9 ng/μg。本批标准物质每瓶1.0 g,使用时最小取样量为100 mg,可用于对转基因大豆MON89788进行定性和定量检测,以及转基因大豆MON89788特异性检测方法的评价和实验室质量控制。本研究为我国转基因检测基体标准物质的研制提供了理论基础和技术支持。

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

In 2009, the genetically modified (GM) rice event TT51-1 with an engineered insect resistance trait became the first GM rice event to be granted certification for safe production in China, and its derivative lines Bt 63 and Huahui No.1 are expected to be commercialized soon. The development of certified reference material (CRM) for TT51-1 is necessary to monitor and inspect the TT51-1 event and its derivates. In this work, we developed four matrix-based TT51-1 rice CRMs (TT51-1a, TT51-1b, TT51-1c, and TT51-1d) with different TT51-1 mass fraction ratios by blending seed powders of homozygous TT51-1 and its recipient cultivar Minghui 63. The between-bottle homogeneity and the within-bottle homogeneity were tested, and good results were obtained. The potential degradation during transportation and shelf life were evaluated, and demonstrated an expiration period of at least 36 months. The characterization values of the four TT51-1 CRMs based on the mass fraction ratio were 1000.000 ± 51.430 g/kg, 49.940 ± 4.620 g/kg, 9.990 ± 1.110 g/kg, and 4.990 ± 0.620 g/kg, respectively. The characterization values based on the copy number ratio were certified by digital PCR analysis as 97.442 ± 5.253 %, 4.851 ± 0.486 %, 1.042 ± 0.135 %, and 0.556 ± 0.073 %, respectively. These results suggested that the TT51-1 matrix-based CRMs developed are of high quality and can be used as potential calibrators for TT51-1 GM rice inspection and monitoring.

首先分析了全球转基因玉米产业化情况,进而从国内转基因玉米产品消费现状、玉米生产中的技术需求以及我国转基因玉米研发现状等方面论述了我国推进转基因玉米产业化的必要性,并从科学上和管理上分析了推动转基因玉米产业化亟待解决的问题,对转基因研发人和安全管理工作提出了建议,以促进转基因玉米产业化的进程。

In order to view the current status and inspiration on the safety detection technology of genetically modified organisms in European Union, this study was fulfilled on the basis of visiting eight detection agencies for genetically modified organisms(GMO)in the European Union(EU). Several key technical points in GMO detection process and the implementation in EU were elaborately expounded. The specific contents included sampling and preparation methods of samples, loop verification of transgenic detection method and parameter requirements, strategies of sample testing and requirements of laboratory environments, representations of test results and evaluation of uncertainty. Finally, combined with the development status of GMO detection techniques in China, suggestions of carrying out inter-laboratory trial validation of detection method and strengthening quantitative PCR detection technology were proposed.

2008年国家启动转基因植物新品种培育重大专项,同时,开展了转基因产品标准物质研制工作,介绍了转基因产品标准物质的概念和作用、种类及其优缺点、研制现状,对今后的标准物质研制工作的重点进行了展望。

对转基因玉米T25转化体特异性荧光定量PCR方法的关键参数进行了方法验证,选择了7家实验室采用经过验证的方法对3个水平的转基因玉米T25基体标准物质进行协同实验研究.通过比较分析各参与实验室的实验条件、标准曲线的参数,进一步证明该方法室间重复性好.经统计学分析各参与实验室的数据等精度,因此计算各实验室的总平均值做为标准物质的标准值.3个水平的基体标准物质的标准值和扩展不确定度(k=2)分别为1.17％±0.22％、2.17％±0.38％和9.81％±2.30％.

Genetically modified organisms detection reference materials(GMOs detection RMs) used in genetically modified organisms(GMOs) detection are essential for GMOs safety supervision, qualitative and quantitative detection. To date, only a few institutions, such as EU Joint Research Centre(Institute for Reference Materials and Measurements, IRMM) and American Oil Chemists' Society(AOCS), have developed certified reference material(CRM) for GMOs testing. In China, research and development of GMOs detection RM are still at the initial stage. Based on the price of CRMs on GMOs testing is very expensive, and the available of CRMs for GMOs can not meet requirements in China, the funding agency in China supported a research project named Study on RMs for GMOs which aims to develop CRMs for GMOs detection used in Chinese GMOs safety supervision requirements. This paper presented overview on production process and value characterization pattern of current existing RMs based on matrix, genomic DNA and plasmid DNAs. We also addressed the problems in development of RMs of GMOs detection in this paper. This review can provide a useful reference for production of Chinese RMs of GMOs detection, and theoretical basis for value characterization and uncertainty evaluation of RMs.

The accuracy,reliability and comparability of genetically modified(GM) detection result can not be ensured due to the lack of GM reference material.In this paper,the selected genetically modified(GM) maize(Zea mays) line NK603 and its recipient were grinded and the particle size was about 100 μm.Three different levels of matrix reference materials were gravimetrically prepared.The homogeneity and stability were checked by quantitative real time PCR.The result showed the matrix reference materials were homogenous and stable under the 4℃ during one year's stock.The property values determined by gravimetric method and their expanded uncertainties(k=2) of three matrix GM reference material were(0.50±0.10)%,(1.00±0.35)% and(5.00±0.35)%,respectively.Seven different laboratories were chosen to validate the property values of the GM maize reference materials and the result showed that it was agreed well with the value determined by the gravimetrical method for each level.The uncertainties of the GM reference materials were comparable with those international GM reference material.Thus,the GM maize reference materials can be applied for the GM maize quantitative detection to ensure the detection result accurate and reliable.

Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM) was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

This paper describes the worldwide production,characterization and application status of genetically modified organism(GMO) detection reference materials(RMs).The different "value" expression patterns and technical approaches of current GMO detection RMs were systematically compared.The advantages and disadvantages of each "value" expression pattern were also elucidated.According to the comparison results,it was suggested that the most suitable pattern of "value" expression and technical approaches of RMs for GMO safety supervision in China.These will provide references for the establishment of characterization system and production and application platform for GMO detection RMs.

The testing institute system for the bio-safety of agricultural GMOs is an important component of the technical supporting system for the bio-safety control of agricultural GMOs.The construction of testing institute system was systematically reviewed from the overall layout,conditions construction,quality certification,operating mode and testing ability,respectively.The roles of testing institute system in administrative management,safety assessment,technical standards development,and risk communication of agricultural GMOs were comprehensively summarized.The shortcomings of the testing institute system in regional location,quality and legal awareness and operative efficiency and its possible reasons were analyzed in-depth.Finally,four strategies and recommendations were proposed focus on consummating the layout,enhancing the quality and legal awareness,improving operational efficiency and building network system.