Development and Characterization of Pure Matrix Reference Materials for Detection of Transgenic Soybean (Glycine max) MON89788
LI Jun1, LI Xia-Ying2, LI Liang3, SONG Gui-Wen2, SHEN Ping2, ZHANG Li1, ZHAI Shan-Shan1, LIU Fang-Fang3, WU Gang1, ZHANG Xiu-Jie2,*, WU Yu-Hua1,*
1 Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs / Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China; 2 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China; 3 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract：The reference materials (RMs) for genetically modified organisms (GMOs) detection are essential for ensuring the accuracy, reliability and traceability of test results. Among different types of RMs, the pure powder RMs has the characteristics of easy-preparation and reliable property value, therefore, to become the most commonly used RMs for GMO quantification. In this study, the homozygous seeds of transgenic soybean (Glycine max) MON89788 were used as raw materials to develop pure matrix RMs through the steps of raw material identification, grinding, particle size measurement, water content measurement and initial homogeneity test. Particals less than 200 μm in this batch of RMs accounted for more than 80% of total powders, and the water content was measured to be (4.53±0.03)%, less than the acceptable level of 10%. A total of 400 bottles of RMs were developed by filling 1 g powder in bottles. 15 bottles were randomly selected from the whole batch of RMs to evaluate the homogeneity by MON89788 and Lectin qRT-PCR methods, the F-test results showed that the RMs powder was well-homogenized among and within bottles. The minimum sampling amount was determined to be as low as 100 mg according to the standard of DNA extraction together with the homogeneity test results. For short-term stability study, the RMs were evaluated by analyzing 3 bottles stored at 4, 25, 37 and 60 ℃ for 1, 2 and 4 weeks. For long-term stability, the RMs were evaluated by analyzing 3 bottles stored at 4 ℃ and -20 ℃ for 1, 2, 4 and 6 months. The property value of RMs did not show obvious changes with the extension of transportation and storage time. A t test results indicated that the slopes of regression curves (β1) were not significantly different from zero under the transportation and storage conditions at the 95% confidence level. The stability test indicated that the RMs could be transported at room temperature for one month and the long-term stability was up to 6 months. The property value of this batch of RMs was defined as the copy number ratio of transgenic DNA to total DNA (copy/copy), which was collaboratively characterized by 9 laboratories using MON89788/Lectin duplex digital PCR. Statistical analysis of the characterization data revealed no outlier and a nominal distribution of the data. The calculated average of all independent measurement results was assigned as the certified value. The certified value of DNA copy number ratio was calculated to be 1.00. The uncertainty of the RMs consisted of uncertainty components from characterization, homogeneity, and stabiltiy during short-term and long-term storage. The expanded uncertainty was estmiated to be 0.07 by combining the 3 components with a coverage factor k (k=2 at 95% confidence level). The property value was certified to be 1.00 with an extended uncertainty of 0.07. This batch of RMs can be used for the qualitative and quantitative detection of GM soybean MON89788, as well as the evaluation of MON89788-specific methods and laboratory quality control.
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