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作物学报 ›› 2020, Vol. 46 ›› Issue (4): 473-483.doi: 10.3724/SP.J.1006.2020.93047

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

转基因玉米MIR604基体标准物质研制

李俊1,李亮2,李夏莹3,宋贵文3,沈平3,张丽1,翟杉杉1,柳方方2,吴刚1,张秀杰3,*(),武玉花1,*()   

  1. 1 中国农业科学院油料作物研究所 / 农业农村部油料作物生物学与遗传育种重点实验室, 湖北武汉 430062
    2 中国农业科学院生物技术研究所, 北京 100081
    3 农业农村部科技发展中心, 北京 100025
  • 收稿日期:2019-08-28 接受日期:2019-12-26 出版日期:2020-04-12 网络出版日期:2020-01-15
  • 通讯作者: 张秀杰,武玉花
  • 作者简介:E-mail: lijuner@163.com, Tel: 027-86711573
  • 基金资助:
    本研究由国家转基因生物新品种培育重大专项(2016ZX08012003);国家自然科学基金项目资助(31601581)

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 Published:2020-04-12 Published online:2020-01-15
  • Contact: Xiu-Jie ZHANG,Yu-Hua WU
  • 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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摘要：

转基因生物安全监管是生物技术产业健康发展的保障, 而转基因检测标准物质是进行转基因监管检测的物质基础, 缺少标准物质就难以保证检测数据的准确性、可靠性和可比性。转基因玉米MIR604是我国批准进口用作加工原料的转基因品种, 对其安全监管亟须制备标准物质。本项目通过对转基因玉米MIR604种子进行原材料鉴定、研磨、过筛、含水量测定等步骤, 制备出转基因玉米MIR604纯品基体标准物质。均匀性检验和稳定性检验结果表明, 本批标准物质在瓶内和瓶间均具有良好的均匀性, 可在常温下运输1个月, 长期稳定性达到6个月, 量值稳定。本批标准物质的特性量值为转基因DNA与总DNA的拷贝数比值, 由9家实验室采用MIR604/Adh1二重数字PCR联合定值, 量值为0.50。充分评估了标准物质定值结果的各个不确定度分量, 合成扩展不确定度为0.06 (copy/copy)。本批标准物质规格为1 g 瓶 -1, 使用时最小取样量为100 mg, 可用于转基因玉米MIR604的安全监管和定量标识、以及实验室质量控制等领域。

关键词: 转基因玉米MIR604, 基体标准物质, 数字PCR, 实时荧光PCR, 联合定值

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

表1

引物探针信息表"

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

图1

鉴定转基因种子基因型的原理示意图 灰白矩形表示受体基因组DNA, 带白斑点的黑色矩形表示外源插入序列, 箭头表示引物位置, 加粗黑线表示探针位置。"

图2

杂合种子的扩增曲线图"

图3

均匀性初检样品的量值测定结果"

图4

均匀性检验样品的测试结果"

表2

均匀性检测测试数据的方差分析结果"

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

表3

标准物质拷贝数比值的短期稳定性检验结果"

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

表4

标准物质拷贝数比值长期稳定性检验结果"

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

表5

转基因MIR604玉米种子粉末标准物质转基因和内标基因比值联合定值数据"

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

表6

标准物质标准值的不确定度分量及合成"

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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