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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (5): 1059-1070.doi: 10.3724/SP.J.1006.2022.13025

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

Development of genetically modified maize (Zea mays L.) NK603 matrix reference materials

SHAN Lu-Ying1(), LI Jun2, LI Liang3, ZHANG Li2, WANG Hao-Qian1, GAO Jia-Qi3, WU Gang2, WU Yu-Hua2,*(), ZHANG Xiu-Jie1,*()   

  1. 1Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China
    2Oil Crops Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062, Hubei, China
    3Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-03-19 Accepted:2021-09-09 Online:2022-05-12 Published:2021-10-11
  • Contact: WU Yu-Hua,ZHANG Xiu-Jie E-mail:2581063925@qq.com;wuyuhua@oilcrops.cn;zhxj7410@sina.com
  • Supported by:
    National Major Project for Developing New GM Crops(2016ZX08012003)

Abstract:

The implementation of genetically modified organisms (GMO) safety management and labeling system requires standardized testing methods and reference materials (RMs). Furthermore, the RMs are the guarantee of obtaining accurate, reliable and comparable testing results. At present, genetically modified (GM) maize (Zea mays L.) NK603 has been approved to be imported as processing raw material in China, and its safety supervision urgently needs to prepare RMs. In this study, the heterozygous seeds of GM maize NK603 and the non-GM maize counterpart after identification were well milled and then gravimetrically mixed to prepare three levels of powder RMs according to the mass fractions of 60.0, 100.0, and 1000 mg g-1. The homogeneity and stability test by duplex ddPCR method showed that the RMs of GM maize NK603 were homogenous and stable for not less than six months. The property values were collaboratively characterized by eight laboratories using duplex ddPCR method. The certified values together with their expanded uncertainties were (2.75±0.26)%, (4.68±0.39)%, and (51.8±3.7)%, respectively. The minimum sample intake was determined to be 100 mg. This batch of RMs can be used for the qualitative and quantitative detection of NK603 event in food and feed, as well as for the evaluation of GM maize NK603-specific assays and laboratory quality control.

Key words: genetically modified maize NK603, reference materials, ddPCR, real-time fluorescent quantitative PCR, collaborative characterization

Table 1

Primer and probe information"

靶标
Target
引物/探针名称Primer/probe name 序列
Primer sequence (5°-3°)
产物大小Amplicon size (bp) 参考文献
Reference
NK603插入位点
NK603 insert site
603CH-F GGAGACAAGAAGGGGAGGAGGTAAACAGAT 267 本研究
This study
603CH-R GGCAGGGTGTTGTTGTCCATTTTATGG
NK603 NK603F ATGAATGACCTCGAGTAAGCTTGTTAA 108 [12]
NK603R AAGAGATAACAGGATCCACTCAAACACT
NK603 P TGGTACCACGCGACACACTTCCACTC
zSSIIb zSSIIbF CGGTGGATGCTAAGGCTGATG 88 [13]
zSSIIbR AAAGGGCCAGGTTCATTATCCTC
zSSIIbP TAAGGAGCACTCGCCGCCGCATCTG
TC1507 TC1507F TAGTCTTCGGCCAGAATGG 58 [14]
TC1507R CTTTGCCAAGATCAAGCG
TC1507P TAACTCAAGGCCCTCACTCCG
MON863 MON863F TGTTACGGCCTAAATGCTGAACT 84 [15]
MON863R GTAGGATCGGAAAGCTTGGTAC
MON863P TGAACACCCATCCGAACAAGTAGGGTCA
GA21 GA21F CTTATCGTTATGCTATTTGCAACTTTAGA 112 [16]
GA21R TGGCTCGCGATCCTCCT
GA21P CATATACTAACTCATATCTCTTTCTCAACAGCAGGTGGGT
MON88017 MON88017F GAGCAGGACCTGCAGAAGCT 95 [17]
MON88017R TCCGGAGTTGACCATCCA
MON88017P FAM-TCCCGCCTTCAGTTTAAACAGAGTCGGGTRA

Fig. 1

Identification of transgenic maize NK603 and non-GM maize by qRT-PCR A: identification of exclusivity of GM (genetically modified) maize NK603; B: identification of exclusivity of non-GM maize NK603."

Fig. 2

Purity test of GM (genetically modified) NK603 maize"

Fig. 3

Electrophoresis detection for genotype identification of GM (genetically modified) maize NK603 1-4 and 9 were amplified with NK603 receptor insertion site primer 603CH-F/R; 5-8 and 10 were amplified with NK603 event specific primer NK603F/R."

Fig. 4

Electrophoresis of the extracted genomic DNA A: kit method; B: CTAB method; 1-9: non-GM (genetically modified) maize; 10-18: GM maize NK603; N: blank control; M: DNA marker DL1000."

Table 2

Genomic DNA extraction rates (μg 100 mg-1)"

样品
Sample
转基因玉米
GM maize NK603
非转基因玉米受体
Non-transgenic receptor maize
1 12.12 11.48
2 12.30 12.24
3 12.00 11.94
4 12.84 13.01
5 13.14 13.20
6 12.42 12.84
7 11.76 12.36
8 12.18 12.12
9 12.60 12.68
t检验t-test 0.64
平均抽提效率比
Average extraction ratio (%)
99.5

Table 3

Weight ratio of the reference materials (RMs) in GM (genetically modified) maize NK603a"

重复
Repetitive
m1 (g) m2 (g) Δm1 (g) Δm2 (g) m (g) 转基因含量
MF (mg g-1)
1 12.005 187.995 0.145 2.181 200.000 60.0
2 12.004 187.994 0.145 2.181 199.998 60.0
3 12.006 187.995 0.145 2.181 200.001 60.0
4 12.005 187.996 0.145 2.181 200.001 60.0
5 12.004 187.996 0.145 2.181 200.000 60.0
6 12.005 187.995 0.145 2.181 200.000 60.0
平均值Mean 12.005 187.995 0.145 2.181 200.000 60.0

Table 4

Weight ratio of the reference materials (RMs) in GM (genetically modified) maize NK603b"

重复
Repetitive
m1 (g) m2 (g) Δm1 (g) Δm2 (g) m (g) 转基因含量
MF (mg g-1)
1 25.011 224.999 0.303 2.610 250.010 100.0
2 25.010 225.001 0.303 2.610 250.011 100.0
3 25.010 224.998 0.303 2.610 250.008 100.0
4 25.012 224.999 0.303 2.610 250.011 100.0
5 25.011 225.001 0.303 2.610 250.012 100.0
6 25.012 225.002 0.303 2.610 250.014 100.0
平均值Mean 25.011 225.000 0.303 2.610 250.011 100.0

Fig. 5

Homogeneity test of GM (genetically modified) maize NK603 powder RMs"

Table 5

Homogeneity analysis of homogeneity test data of GM (genetically modified) maize NK603 powder RMs"

样品
Sample
差来源
Soruces of variation
差方和
Q
自由度
df
方差
S
统计量
F
F0.05 (14,30) 比较结果
Test data
结论
Result
NK603a 瓶间Among bottles 0.43 14 0.031 1.93 2.04 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 0.48 30 0.016
NK603b 瓶间Among bottles 1.18 14 0.084 1.76 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 1.43 30 0.048
NK603c 瓶间Among bottles 16.87 14 1.200 0.85 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 42.57 30 1.420

Table 6

Short-term stability of copy number ratio of NK603 powdered RMs"

项目
Item
NK603a NK603b NK603c
25℃ 37℃ 60℃ 25℃ 37℃ 60℃ 25℃ 37℃ 60℃
0 d 2.84 2.84 2.84 4.62 4.74 4.80 51.68 51.68 51.68
3 d 2.84 2.89 2.92 4.75 4.65 4.62 51.24 51.33 51.78
7 d 2.88 2.74 2.79 4.63 4.79 4.59 53.01 52.11 50.41
14 d 2.72 2.66 2.82 4.84 4.77 4.62 51.32 52.12 51.79
平均值Mean 2.82 2.78 2.84 4.71 4.74 4.66 51.8108 51.81 51.41
斜率(β1) -0.0082 -0.0150 0.0039 0.0121 0.0052 -0.0100 -0.0022 0.0458 -0.0060
与斜率相关的不确定度s(β1) 0.006 0.005 0.006 0.008 0.006 0.009 0.096 0.030 0.078
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) 0.0237 0.0226 0.0270 0.0350 0.0265 0.0375 0.4135 0.1300 0.3364
稳定性判断
Stability judgment
|β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1)
判断结果
Conclusion
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable
稳定
Stable

Table 7

Long-term stability of copy number ratio of NK603 powder RMs"

项目
Item
NK603a NK603b NK603c
4℃ -20℃ 4℃ -20℃ 4℃ -20℃
0个月0 month 2.76 2.80 4.84 4.71 50.85 51.20
1个月1 month 2.83 2.86 4.75 4.76 50.61 51.36
2个月 2 months 2.81 2.77 4.78 4.79 50.67 51.73
4个月 4 months 2.74 2.86 4.81 4.83 51.86 50.69
6个月 6 months 2.81 2.87 4.82 4.77 51.59 51.64
平均值Mean 2.79 2.83 4.80 4.77 51.12 51.32
斜率(β1) 0.0011 0.0109 0.0023 0.0112 0.1910 0.0085
与斜率相关的不确定度s(β1) 0.0092 0.0093 0.0082 0.0083 0.0812 0.0984
t0.95,n-2 3.1824 3.1824 3.1824 3.1824 3.1824 3.1824
t0.95,n-2×s(β1) 0.0292 0.0296 0.0261 0.0265 0.2584 0.3132
稳定性判断Stability judgment |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1)
判断结果Conclusion 稳定Stable 稳定Stable 稳定Stable 稳定Stable 稳定Stable 稳定Stable

Table 8

Statistics of the characterized value of NK603 powder RMs"

参数
Parameter
结果Result
NK603a NK603b NK603c
标准值Certified value (copy/copy) (%) 2.75 4.68 51.80
组间SD SD between groups 0.03 0.06 0.63
组间RSD RSD between groups (%) 1.19 1.31 1.21

Table 9

Components and combination of uncertainty for the copy number ratio of NK603 powder RMs"

标准物质
名称
RMs name
标准值Certified value Y (%) 定值相对
不确定度
urel.c
均匀性相对不确定度urel,bb 短期稳定性相对不确定度urel,ss 长期稳定性相对不确定度urel,ls 相对标准不确定度urel,CRM 相对扩展不确定度Urel,CRM UCRM (k=2) (%)
NK603a 2.75 0.0202 0.025 0.028 0.020 0.047 0.094 0.26
NK603b 4.68 0.0210 0.024 0.025 0.011 0.041 0.082 0.39
NK603c 51.80 0.0203 0.007 0.026 0.010 0.035 0.071 3.70
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