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
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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