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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (4): 473-483.doi: 10.3724/SP.J.1006.2020.93047

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Development of genetically modified maize MIR604 matrix reference materials

LI Jun1,LI Liang2,LI Xia-Ying3,SONG Gui-Wen3,SHEN Ping3,ZHANG Li1,ZHAI Shan-Shan1,LIU Fang-Fang2,WU Gang1,ZHANG Xiu-Jie3,*(),WU Yu-Hua1,*()   

  1. 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, Hubei, China
    2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China
  • Received:2019-08-28 Accepted:2019-12-26 Online:2020-04-12 Published:2020-01-15
  • Contact: Xiu-Jie ZHANG,Yu-Hua WU E-mail:zhxj7410@sina.com;wuyuhua@oilcrops.cn
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX08012003);the National Natural Science Foundation of China(31601581)

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Abstract:

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.

Key words: GM maize MIR604, matrix reference material, digital PCR, Real-time PCR, collaborative characterization

Table 1

List of primer and probe information"

靶标
Target		 引物名称
Primer name		 序列
Sequence (5°-3°)		 产物大小
Amplicon size (bp)		 参考文献
Reference
MIR604 MIR604F GCGCACGCAATTCAACAG 76 Mazzara et al. 2007[10]
MIR604R GGTCATAACGTGACTCCCTTAATTCT
MIR604P AGGCGGGAAACGACAATCTGATCATG
Adh1 ADH-FF3 CGTCGTTTCCCATCTCTTCCTCCT 135 Mazzara et al. 2013[10]
ADH-RR4 CCACTCCGAGACCCTCAGTC
ADH1-MDO AATCAGGGCTCATTTTCTCGCTCCTCA
MON810 MON810F TCGAAGGACGAAGGACTCTAACGT 92 ISO 21570[11]
MON810R GCCACCTTCCTTTTCCACTATCTT
MON810P AACATCCTTTGCCATTGCCCAGC
MON863 MON863F TGTTACGGCCTAAATGCTGAACT 84 Mazzara et al. 2005[12]
MON863R GTAGGATCGGAAAGCTTGGTAC
MON863P TGAACACCCATCCGAACAAGTAGGGTCA
NK603 NK603-F ATGAATGACCTCGAGTAAGCTTGTTAA 108 Mazzara et al. 2005[13]
NK603-R AAGAGATAACAGGATCCACTCAAACACT
NK603-PR TGGTACCACGCGACACACTTCCACTC
T25 MLD143 ACAAGCGTGTCGTGCTCCAC 102 Mazzara et al. 2013[14]
MDB551 GACATGATACTCCTTCCACCG
TM016 TCATTGAGTCGTTCCGCCATTGTCG
TC1507 TC1507F TAGTCTTCGGCCAGAATGG 58 Mazzara et al. 2007[15]
TC1507R CTTTGCCAAGATCAAGCG
TC1507P TAACTCAAGGCCCTCACTCCG
MIR604插入位点
MIR604 insert site		 MIR604GF GACGCCAGATCACACATG 168 本研究
This study
MIR604GR CACACCTCGTTGCCAAAG
MIR604GP CGTGCCGATCTCGCACCAGCA

Fig. 1

Schematic diagram of identifying genotype of genetically modified seeds The grey rectangles indicate the recipient genomic DNA, the black rectangles with white spots indicate the insert DNA, the arrows indicate the position of the primers, and the bold black lines indicate the position of the probes."

Fig. 2

Amplification curves of heterozygous seed"

Fig. 3

Measured values of initial test samples for homogeneity"

Fig. 4

Measurement results for homogeneity test of reference materials"

Table 2

Variance analysis of homogeneity test data"

项目
Item		 分析结果
Test data
测量平均值Mean 0.5
组间差方和Q1 0.013
组间自由度V1 14
组间方差S12 0.000922
组内差方和Q2 0.019
组内自由度V2 30
组内方差S22 0.000649
统计量F 1.42
临界值F0.05(14, 30) 2.04
比较Comparison F<F0.05(14, 30)
结论Conclusion 均匀Homogenous

Table 3

Short-term stability of copy number ratio"

时间
Time		 贮存温度 Storage temperature
4℃ 25℃ 37℃ 60℃
0周 0 week 0.499 0.493 0.500 0.489
1周 1 week 0.507 0.514 0.509 0.518
2周 2 weeks 0.510 0.504 0.488 0.500
4周 4 weeks 0.493 0.498 0.513 0.501
平均值 Mean 0.502 0.502 0.503 0.502
斜率Slope (β1) -0.002 -0.0004 0.002 0.001
与斜率相关的不确定度Uncertainty associated with slope s(β1) 0.0029 0.0037 0.0043 0.0049
t0.95,n-2 4.303 4.303 4.303 4.303
t0.95,n-2×s(β1) 0.013 0.016 0.018 0.021
稳定性判断Conclusion 稳定Stable 稳定Stable 稳定Stable 稳定Stable

Table 4

Long-term stability of copy number ratio"

时间
Time		 4℃条件下测量值
Measured value at 4℃		 -20℃条件下测量值
Measured value at -20℃
0个月 0 month 0.496 0.506
1个月 1 month 0.486 0.494
2个月 2 months 0.508 0.495
4个月 4 months 0.492 0.484
6个月 6 months 0.506 0.506
平均值Mean 0.498 0.497
斜率Slope (β1) 0.003 -0.001
与斜率相关的不确定度Uncertainty associated with slope s(β1) 0.0031 0.0034
t0.95,n-2 3.182 3.182
t0.95,n-2×s(β1) 0.010 0.011
稳定性判断Conclusion 稳定Stable 稳定Stable

Table 5

Data of collaborative characterization for the copy number ratio of MIR604 powdered reference materials"

实验室编号
Lab. no.		 重复1
Rep.1		 重复2
Rep.2		 重复3
Rep.3		 重复4
Rep.4		 重复5
Rep.5		 重复6
Rep.6		 重复7
Rep.7		 重复8
Rep.8
1 0.5029 0.5095 0.5110 0.5070 0.5109 0.5042 0.4968 0.5087
2 0.5088 0.4990 0.5000 0.4896 0.5191 0.5064 0.5183 0.5021
3 0.5135 0.4941 0.5070 0.5030 0.5010 0.4943 0.5018 0.4861
4 0.5141 0.5119 0.4903 0.5070 0.4949 0.4829 0.4990 0.5128
5 0.5011 0.4844 0.5072 0.4898 0.5053 0.4927 0.5077 0.4947
6 0.5094 0.5170 0.5064 0.5090 0.4948 0.5126 0.4861 0.4965
7 0.5000 0.4859 0.5132 0.4909 0.5167 0.5000 0.5082 0.4917
8 0.4990 0.5114 0.4858 0.4951 0.4981 0.5134 0.5028 0.5038
9 0.5143 0.5115 0.4962 0.5028 0.5163 0.5059 0.5091 0.4962
平均值Mean 0.5027
标准差SD 0.001
相对标准差RSD 0.002

Table 6

Components and combination of uncertainty for the copy number ratio of MIR604 powdered reference materials"

标准物质
标准值
Certified value		 定值引入的相对不确定度uc 均匀性引入的相对不确定度ubb 稳定性引入的相对不确定度us 相对不确定度
uCRM		 扩展不确定度
UCRM (k = 2)
A类uA B类uB 短期稳定性uss 长期稳定性uls
0.50 0.002 0.014 0.019 0.030 0.038 0.054 0.06
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