Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (8): 1444-1455.doi: 10.3864/j.issn.0578-1752.2023.08.002


Development of A Set of Matrix Reference Materials in Different Mass Fractions of Genetically Modified Maize MON87427

LI Jun1(), SHAN LuYing2, XIAO Fang1, LI YunJing1, GAO HongFei1, ZHAI ShanShan1, WU Gang1, ZHANG XiuJie2(), WU YuHua1()   

  1. 1 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of agricultural genetically modified organism traceability, Ministry of Agriculture and Rural Affairs, Wuhan 430062
    2 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025
  • Received:2022-10-28 Accepted:2022-12-08 Online:2023-04-16 Published:2023-04-23


【Objective】The GMO (genetically modified organism) reference materials (RMs) are the material basis for GMO safety supervision and labeling policy implementation. During GMO detection the utilization of RMs guarantees the traceability of quantitative results within laboratory and the comparability of quantitative results between laboratories. GM (genetically modified) maize MON87427 has been approved to be imported as raw material in China, it’s urgent to develop certified reference materials (CRMs) for safety supervision and quantification. 【Method】 The GM maize MON87427 hybrid seeds and non-GM counterparts provided by the developer were used as raw materials to perform washing, drying, freeze-grinding, particle size measurement, and moisture content measurement, sequentially. The matrix RMs of MON87427a, MON87427b and MON87427c were produced by blending the seed powder of the GM maize MON87427 and a non-GM counterpart in GMO mass fractions of 60.0 mg·g-1, 99.5 mg·g-1 and 1 000.0 mg·g-1 on a dry basis. The real-time quantitative PCR was used to conduct an initial assessment of homogeneity before packing. The MON87427/zSSIIb duplex digital PCR (ddPCR) was used to evaluate the homogeneity and stability of the RMs as well as the collaborative characterization by 8 qualified laboratories. The data processing of homogeneity, stability, collaborative characterization together with uncertainty evaluation of RMs were carried out according to the standard "General and Statistical Principles for Characterization of Reference Materials" (JJF 1343). 【Result】 This batch of RMs contains three RMs of MON87427a, MON87427b, and MON87427c in different mass fractions, with more than 80% of particle size of less than 200 μm and less than 5% of moisture content. The RMs were packed in brown glass bottles, nitrogen flushed before capping, bottling amount was not less than 1.0 g/bottle, a total of 400 bottles were produced for each mass fraction RM. The calculated F values of the homogeneity test were all less than the critical value of F0.05 (14, 30) (2.04) for the three RMs, displaying good homogeneity within and between bottles, and the minimum intake was determined to be 100 mg. The RMs can be stored stably at 25℃, 37℃, and 60℃ for 14 days, the property value of the RMs does not change significantly after 14 days of transportation at room temperature; the long-term stability can reach 12 months at 4℃ and -20℃; The property value of the samples taken from the same bottle of RM after 5 opening-capping cycles, does not deviate significantly from that of the first taken sample. The collaborative characterization data by eight qualified laboratories displayed normal distribution without outliers and outlying standard deviations. The standard value and expanded uncertainty of MON87427a, MON87427b, Mon87427c were certified to be (2.92±0.44)%, (4.89±0.57)%, (52.1±3.4)%. 【Conclusion】The developed GM maize MON87427 matrix RMs in different mass fractions have good homogeneity, and can be stably transported and stored. This batch of RMs meets the requirements of qualitative and quantitative detection of MON87427 event, providing reliable CRMs for the safety supervision and implementation of quantitative labeling policy for GMO-derived products.

Key words: genetically modified maize MON87427, matrix reference material, duplex digital PCR, property value, uncertainty

Table 1

List of primer and probe of PCR and amplicon size"

PCR type
Sequence of primer/probe (5′-3′)
Amplicon size (bp)
Real-time PCR
Conventional PCR
Insert site

Table 2

Mass of GM powder and non-GM powder weighed for preparation MON87427a and MON87427b"

Reference materials
拟称取质量 Proposed weighing mass (g) 实际称取质量 Practical weighing mass (g) 含水量 Water content (g) 转基因含量
GMO content (mg·g-1)
Total mass
GM powder
Non-GM powder
Total mass
GM powder
Non-GM powder
GM powder
Non-GM powder
MON87427a 450 26.995 423.005 450.002 27.005 422.997 0.292 4.653 60.02
MON87427b 450 44.992 405.008 450.000 44.778 405.222 0.484 4.457 99.52

Table 3

Initial test of MON87427 reference materials homogeneity"

Reference materials
MON87427a MON87427b MON87427c
12 h 18 h 24 h 12 h 18 h 24 h 12 h 18 h 24 h
平均值 Mean 3.31 3.25 3.33 5.44 5.23 5.24 51.22 51.84 52.20
标准差 SD 0.21 0.12 0.08 0.63 0.35 0.17 1.77 1.34 2.09
相对标准差 Relative SD 6.40 3.54 2.51 11.55 6.68 3.26 3.45 2.59 4.01
统计量F Statistic F 1.60 1.73 2.18 2.17 0.56 0.74 1.31 2.32 1.89
临界值 F0.05(8,18) 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51 2.51
结论 Conclusion 均匀 Homogeneous 均匀 Homogeneous 均匀 Homogeneous

Table 4

ANOVA results of MON87427 RM homogeneity"

Variance source
Sum of squares of deviation
Degree of freedom
Statistic F
F0.05 (14,30) 比较结果
MON87427a 瓶间 Between-vial 0.25 14 0.018 1.70 2.04 F<F0.05 (14,30) 均匀
瓶内 Within-vial 0.31 30 0.010
MON87427b 瓶间 Between-vial 0.56 14 0.040 1.40 F<F0.05 (14,30) 均匀
瓶内 Within-vial 0.85 30 0.028
MON87427c 瓶间 Between-vial 11.97 14 0.855 1.63 F<F0.05 (14,30) 均匀
瓶内 Within-vial 15.75 30 0.525

Table 5

Evaluation of short-term stability of MON87427 reference materials (copy/copy)"

<BOLD>R</BOLD>eference materials
MON87427a MON87427b MON87427c
25℃ 37℃ 60℃ 25℃ 37℃ 60℃ 25℃ 37℃ 60℃
平均值Mean 2.77 2.76 2.87 4.80 4.63 4.63 49.73 50.53 50.14
斜率(β1)Slope -0.0122 0.0085 0.0031 -0.0095 -0.0127 -0.0045 -0.0666 0.0650 -0.0443
与斜率相关的不确定度s(β1) 0.0092 0.0026 0.0129 0.0062 0.0051 0.0069 0.0284 0.0656 0.0882
t0.95,n-2 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027
t0.95,n-2* s(β1) 0.0397 0.0111 0.0556 0.0268 0.0218 0.0297 0.1224 0.2823 0.3793
稳定性判断 Comparison |β1| <t0.95n-2·s(β1)

Fig. 1

The change trend of GMO content of GM maize reference materials MON874277a, MON87427b, MON87427c stored at 4℃, -20℃ for 0, 1, 2, 4, 6 and 12 months, respectively"

Table 6

Evaluation of opening-closing stability of MON87427 reference materials"

<BOLD>R</BOLD>eference materials
MON87427a MON87427b MON87427c
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
平均值Mean 2.68 2.66 2.71 2.68 2.67 4.52 4.43 4.43 4.52 4.35 51.6 51.1 52.56 51.9 52.14
tt value 0.43 -0.54 -0.08 0.28 0.84 0.90 -0.04 2.04 0.51 -0.96 -0.27 -0.49
t(0.05,6) 2.45 2.45 2.45
结论 Conclusion 一致 Consistent 一致 Consistent 一致 Consistent

Statistical analysis of characterization data of MON87427 reference materials"

参数Parameter MON87427a MON87427b MON87427c
标准值 Certified value (copy/copy, %) 2.92 4.89 52.14
组间标准差(A类不确定度)SD(A type uncertainty) 0.06 0.11 0.68
组间相对标准差(A类相对不确定度)RSD (%) (A type relative uncertainty) 2.20 2.34 1.30

Table 8

Property values and related uncertainty components of MON87427 matrix reference materials"

<BOLD>R</BOLD>eference materials
UCRM (k=2)
MON87427a 2.92 0.0237 0.0176 0.0465 0.0508 0.0749 0.1499 0.44
MON87427b 4.89 0.0250 0.0132 0.0181 0.0471 0.0578 0.1157 0.57
MON87427c 52.14 0.0157 0.0065 0.0082 0.0264 0.0324 0.0649 3.40

Fig. 2

Flow chart of the development of mass fraction reference materials"

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