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| Development of Genomic DNA Reference Materials for Genetically Modified Soybean (Glycine max) 'DBN9004' |
| ZHAO Xin1, LI Rui-Huan1, ZHANG Hua2, XU Chao1, QI Xin1, CHEN Zi-Yan2, LIU Na1, WANG Cheng1, LAN Qing-Kuo1, WANG Yong1,* |
1 Tianjin Academy of Agricultural Sciences, Tianjin 300384, China;
2 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100176, China |
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Abstract In the research and standardization of detection methods for genetically modified (GM) products, qualitative and quantitative detection, implementation of labeling regulations, and safety supervision, certified reference materials (CRMs) for GM detection are indispensable. GM soybean (Glycine max) 'DBN9004', a herbicide-tolerant soybean line independently developed in China, obtained a biosafety certificate for production and application in 2020. There is an urgent need to develop certified reference materials for its safety supervision and detection. In this study, using homozygous GM soybean 'DBN9004' and non-transgenic receptor soybean 'Jack' as raw materials, the leaves were frozen, ground, and genomic DNA was extracted. The reference materials DBN9004a and DBN9004c were prepared by weighing and mixing to achieve DBN9004/Lectin copy number ratios of 100% and 3%, respectively. Each concentration of the standard substances was aliquoted into 500 tubes. Using the droplet digital PCR method, according to General Principles and Statistical Principles for Certification of Reference Materials (JJF 1343), the homogeneity evaluation, stability evaluation, processing of joint certification data, and uncertainty assessment of reference materials with 2 concentrations were carried out. The results showed that, the homogeneity test results for both materials were below the critical value F0.05 (14,30)(2.04), indicating good intra- and inter-unit homogeneity. The reference materials can be stored and transported stably for 10 d at temperatures below 37 ℃, and long-term stability at -20 ℃ was confirmed for up to 9 months. After 10 freeze-thaw cycles. The collaborative study involving 9 laboratories showed no outliers in the copy number concentration and ratio measurements, with normal distribution and equal precision. The copy number concentrations and expanded uncertainties for DBN9004a and DBN9004c were (4.43±0.38)×104 and (6.32±0.65)×102 copies/μL, respectively. The DBN9004/Lectin copy number ratios and expanded uncertainties were (98.1±9.8)% and (3.08±0.32)%, respectively. The developed gradient genomic DNA reference materials for GM soybean DBN9004 exhibit good homogeneity and stability during transport and storage. They meet the requirements for qualitative and quantitative detection of GM soybean 'DBN9004' and provide reliable certified reference materials for the safety supervision and implementation of quantitative labeling regulations for GM soybean 'DBN9004'.
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Received: 08 April 2025
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Corresponding Authors:
* Corresponding author, wytaas@126.com
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[1] 董莲华, 赵正宜, 李亮, 等. 2012. 转基因植物标准物质研究进展[J].农业生物技术学报, 20(02):203-210.
(Dong L H, Zhao Z Y, Li L, et al.2012. Advance in developing genetically modified plant reference material[J]. Journal of Agricultural Biotechnology, 20(02): 203-210.)
[2] 高芳瑞, 王颢潜, 李瑞环, 等. 2022. 转基因检测标准物质研制关键环节质量控制研究[J].中国生物防治学报, 38(05):1125-1134.
(Gao F R, Wang H Q, Li R H, et al.2022. Research on key link and critical control point of GMO testing reference material[J]. Chinese Journal of Biological Control, 38(05): 1125-1134.)
[3] 焦悦, 王智, 张振民, 等. 2022. 基于转基因产品成分低水平混杂问题探究我国转基因阈值制度[J].中国农业科技导报, 24(03): 20-27.
(Jiao Y, Wang Z, Zang Z M, et al.2022. Research on the transgenic threshold system in china based on low-level presence of genetically modified products[J]. Journal of Agricultural Science and Technology, 24(03): 20-27.)
[4] 阚莹, 李红梅. 2012. JJF 1343 - 2012, 标准物质定值的通用原则及统计学原理[S]. 北京: 中国标准出版社, pp. 2-50.
(Kan Y, Li H M.2012. JJF 1343 - 2012, General and Statistical Principles for Characterization of Reference Materials[S]. Beijing: China Standards Press, pp. 2-50.)
[5] 李保茹. 2025. 中国转基因种子的生态安全评估与法律法规监管[J]. 分子植物育种, 23(03): 829-834.
(Li B R.2025. Ecological safety assessment and legal regulation of genetically modified seeds in China[J]. Molecular Plant Breeding, 23(03): 829-834.)
[6] 李俊, 李夏莹, 李亮, 等. 2020. 转基因大豆MON89788纯品粉末标准物质的研制及定值[J].农业生物技术学报, 28(06):1084-1095.
(Li J, Li X Y, Li L, et al.2020. Development and characterization of pure matrix referencematerialsfor detection of transgenic soybean (Glycine max)MON89788[J]. Journal of Agricultural Biotechnology, 28(06): 1084-1095.)
[7] 林鹰, 杨文莉, 周玲艳, 等. 2022. 农业转基因核酸标准物质研究进展[J]. 生物技术通报, 38(08): 52-59.
(Lin Y, Yang W L, Zhou L Y, et al.2022. Research progress in agricultural genetically modified nucleic acid reference materials[J]. Biotechnology Bulletin, 38(08): 52-59.)
[8] 罗阿东, 焦彦朝, 曹云恒, 等. 2012. 转基因大豆检测技术研究进展[J].南方农业学报, 43(03): 290-293.
(Luo A D, Jiao Y C, Cao Y H, et al.2012. Progresses in detection technologies for transgenic soybean[J]. Journal of Southern Agriculture, 43(03): 290-293.)
[9] 文婷婷. 2020. 转基因产品核酸成分标准物质研制关键技术研究[D]. 硕士学位论文, 长春理工大学, 导师: 张晓, pp.56-66.
(Wen T T.2020. Research on key technologies for the development of nucleic acid composition reference materials in genetically modified products[D]. Thesis for M.S., Changchun University of Science and Technology, Supervisor: Zhang X, pp.56-66.)
[10] 许丽, 梁文, 李妍, 等. 2014. 一种质粒DNA标准物质的定值数据统计及不确定度评定[J]. 中国测试, 40(S1): 9-13.
(Xu L, Liang W, Li Y, et al.2014. The data statistic and uncertainty assessment for the quantification of a plasmid DNA reference material[J]. China Measurement & Test, 40(S1): 9-13.)
[11] 徐琳杰, 刘培磊, 李文龙, 等. 2018. 国际转基因标识制度变动趋势分析及对我国的启示[J].中国生物工程杂志, 38(09): 94-98.
(Xu L J, Liu P L, Li W, et al.2018. Long analysis of the trend of international transgenic labeling system and its enlightenment to China[J]. China Biotechnology, 38(09): 94-98.)
[12] 赵新, 刘双, 刘娜, 等. 2022. 耐除草剂大豆'DBN9004'精准定量检测方法的建立[J].农业生物技术学报, 30(12):2446-2455.
(Zhao X, Liu S, Liu N, et al.2022. Establishment of accurate quantitative detection method for herbicide-tolerant soybean (Glycine max) 'DBN9004'[J]. Journal of Agricultural Biotechnology, 30(12): 2446-2455.)
[13] 郑兰. 2017.七种转基因水稻质粒DNA标准物质研制[D]. 硕士学位论文, 上海交通大学, 导师: 王灿华. pp. 57-70.
(Zheng L.2017. Development of seven plasmids reference materials for detection of genetically modified rice[D]. Thesis for M.S., Shanghai Jiao Tong University, Supervisor: Wang C H, pp. 57-70.)
[14] 郑子繁, 柳方方, 刘卫晓, 等. 2020. 数字PCR技术在核酸标准物质研制中的应用[J]. 生物技术进展, 10(06): 579-584.
(Zheng Z F, Liu F F, Liu W X, et al.2020. Application of digital PCR technology in the development of nucleic acid[J]. Current Biotechnology, 10(06): 579-584.)
[15] 卓勤, 杨晓光. 2019. 各国转基因食品标识制度概况分析[C]. 达能营养中心2019年论文汇编: 转基因食品与安全. 中国疾病预防控制中心营养与食品安全所, pp. 21-26.
(Zhuo Q, Yang X G.2019. analysis of the worldwide management for labeling of genetically modified food[C]. Danone Nutritional Center 2019 Papers Compilation: Genetically Modified Food and Safety. Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, pp. 21-26.)
[16] Cheng X G, Li H H, Tang Q L, et al.2024. Trends in the global commercialization of genetically modified crops in 2023[J]. Journal of Integrative Agriculture, 23(12): 3943-3952.
[17] Hindson B J, Ness K D, Masquelier D A, et al.2011. High - throughput droplet digital PCR system for absolute quantitation of DNA copy number[J]. Analytical Chemistry, 83(22): 8604-8610.
[18] Li J, Zhang L, Li L, et al.2020. Development of genomic dna certified reference materials for genetically modified rice Kefeng 6[J]. ACS Omega, 5(34): 21602-21609.
[19] Song X, Wang L, Liu X, et al.2021. Advances in detection technologies for genetically modified soybeans[J]. Journal of Agricultural and Food Chemistry, 69(31): 8895-8904.
[20] Wang J, Wang J, Xu X, et al.2020. Development and application of reference materials for genetically modified detection[J]. Journal of Analytical and Testing, 39(12): 1577-1583.
[21] Yang X, Li N, Huang K, et al.2021. Current situation and development suggestions of genetically modified organism safety management in China[J]. Scientia Agricultura Sinica, 54(17): 3637-3649. |
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