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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (1): 238-248.doi: 10.3724/SP.J.1006.2022.12001

• RESEARCH NOTES • Previous Articles     Next Articles

Effect of 11-year storage of GMO reference material at ultra-low temperature on nucleic acid detection of standard matrix sample of transgenic crop

WANG Wei-Xia(), LAI Feng-Xiang, HU Hai-Yan, HE Jia-Chun, WEI Qi, WAN Pin-Jun, FU Qiang*()   

  1. China National Rice Research Institute, Hangzhou 310006, Zhejiang, China
  • Received:2021-01-12 Accepted:2021-06-18 Online:2022-01-12 Published:2021-07-19
  • Contact: FU Qiang E-mail:wangweixia@caas.cn;fuqiang@caas.cn
  • Supported by:
    Rice Pest Management Research Group of the Agricultural Science and Technology Innovation Program of China Academy of Agricultural Science(CAAS-ASTIP-2016-CNRRI);Fundamental Research Funds for Central Public Welfare Research Institute(2017RG005)

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Abstract:

Reference material is indispensable for the reliability, comparability, and accuracy of transgenic detection results. In this paper, the effects of long-term preservation of matrix reference materials on PCR detection and the stability of low content reference materials were studied. The reproducibility and quantity stability of transgenic reference materials and transgenic samples with different contents were detected by three methods (common qualitative PCR, real-time fluorescent PCR, and droplet digital PCR). The result showed that the transgenic reference material with 0.1% GM content could still be clearly amplified after 9 years of storage, and the Ct value was about 36. The digital PCR revealed that the quantity of GM matrix reference materials remained stable for long storage time. There was no effect on the detection of transgenic elements in sample mixed with soybean, corn and rice powder which stored at -70 to -75℃ for 11 years. In summary, the long-term storage of GM matrix reference materials at low temperature would not affect the detection of each transgenic component and its application as a positive control for transgenic nucleic acid detection.

Key words: genetically modified organism (GMO), matrix reference material, detection of transgenic plants, reference material storage

Table 1

Information of transgenic detection parameters and recommended standards of the retained mixed crop samples"

样品编号
Sample ID		 起存时间
Start date		 存期
Storage		 阳性转基因元件及检测参考标准
Positive transgenic elements and references
S1 2018/10 2年 2 years CaMV 35S[15], FMV 35S[15], bar/pat[16]
S2 2018/10 2年 2 years CaMV 35S, FMV 35S, NOS[15], HPT[17], bar/pat, Bt[18]
S3 2017/10 3年 3 years CaMV 35S, FMV 35S, NOS, HPT, bar/pat, Bt, CP4-EPSPS[19]
S4 2017/10 3年 3 years CaMV 35S, FMV 35S, NOS, bar/pat, Bt, HPT, CP4-EPSPS, NK603[20], TT51-1[21]
S5 2015/10 5年 5 years CaMV 35S, FMV 35S, NOS, CP4-EPSPS
S6 2015/10 5年 5 years CaMV 35S, NOS, bar/pat, CP4-EPSPS
S7 2014/10 6年 6 years CaMV 35S, FMV 35S, NOS, bar/pat, TT51-1, GT73[22], MS8[23], Bt11[24], MON810[25]
S8 2014/10 6年 6 years FMV 35S, T-NOS, bar/pat, MS8, GT73, TT51-1
S9 2009/10 11年 11 years CaMV 35S, T-NOS, CP4-EPSPS, GTS40-3-2[26], MON863[27]
S10 2009/10 11年 11 years CaMV 35S, T-NOS, Bt, HPT, T-CaMV 35S, MON863

Fig. 1

Agarose gel electrophoresis of genomic DNA from a single crop reference material M: Trans2K plus DNA ladder; Lanes 1-13 are the corresponding DNA sample number shown in Table 3. Loading amount was 4.0 μL."

Fig. 2

Electrophoretic image of GMO reference material with content 0.1% detected by event-specific qualitative PCR The events with 0.1% content were matrix reference materials stored nine years at low temperature; M: 100 bp DNA ladder; 1-6: GMO reference material with 0.1% content; 7-9: positive control with content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: blank control; 16-18: PCR blank H2O control."

Fig. 2

Electrophoretic image of GMO reference material with content 0.1% detected by event-specific qualitative PCR The events with 0.1% content were matrix reference materials stored nine years at low temperature; M: 100 bp DNA ladder; 1-6: GMO reference material with 0.1% content; 7-9: positive control with content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: blank control; 16-18: PCR blank H2O control."

Fig. 3

Amplification curves of GMO reference material with GM content 0.1% detected by qRT-PCR The events with 0.1% content were matrix reference materials stored nine years at low temperature; CK+: positive control with content 1.0% (w/w)."

Fig. 3

Amplification curves of GMO reference material with GM content 0.1% detected by qRT-PCR The events with 0.1% content were matrix reference materials stored nine years at low temperature; CK+: positive control with content 1.0% (w/w)."

Table 2

Detection of reference materials with GM content 0.1% stored for nine years by event-specific qRT-PCR"

样品名称
Sample		 模板浓度
DNA concentration (mg L-1)		 Ct值
Ct value		 相对标准差
RSD (%)		 结论
Result		 阳性对照Ct值
Ct value of CK+
Bt11 36.0 36.89±0.53 1.43 阳性Positive 29.32±0.40
Bt176 24.5 34.17±0.09 1.30 阳性Positive 25.39±0.10
MON863 34.5 35.60±0.34 0.95 阳性Positive 30.60±0.19
MON810 28.5 35.05±0.41 1.18 阳性Positive 27.12±0.16
GTS40-3-2 40.0 36.23±0.40 1.11 阳性Positive 30.43±0.44
G6H1 77.5 33.70±0.38 1.12 阳性Positive 27.49±0.52
TC1507 24.5 35.06±0.88 2.52 阳性Positive 30.34±0.49
59122 36.0 36.11±0.42 1.18 阳性Positive 30.25±0.15
MIR604 27.5 35.82±0.77 2.16 阳性Positive 30.31±0.13
GBH119 67.0 31.56±0.15 0.48 阳性Positive 26.98±0.23

Table 2

Detection of reference materials with GM content 0.1% stored for nine years by event-specific qRT-PCR"

样品名称
Sample		 模板浓度
DNA concentration (mg L-1)		 Ct值
Ct value		 相对标准差
RSD (%)		 结论
Result		 阳性对照Ct值
Ct value of CK+
Bt11 36.0 36.89±0.53 1.43 阳性Positive 29.32±0.40
Bt176 24.5 34.17±0.09 1.30 阳性Positive 25.39±0.10
MON863 34.5 35.60±0.34 0.95 阳性Positive 30.60±0.19
MON810 28.5 35.05±0.41 1.18 阳性Positive 27.12±0.16
GTS40-3-2 40.0 36.23±0.40 1.11 阳性Positive 30.43±0.44
G6H1 77.5 33.70±0.38 1.12 阳性Positive 27.49±0.52
TC1507 24.5 35.06±0.88 2.52 阳性Positive 30.34±0.49
59122 36.0 36.11±0.42 1.18 阳性Positive 30.25±0.15
MIR604 27.5 35.82±0.77 2.16 阳性Positive 30.31±0.13
GBH119 67.0 31.56±0.15 0.48 阳性Positive 26.98±0.23

Table 3

Quantitative results of G6H1 and GBH119 with simplex droplet dPCR"

样品含量
Sample
content (%)		 靶标
Target		 拷贝数Copies (20 μL-1) 平均值
Average		 转基因含量
GM content (%)		 相对标准差RSD (%) 偏差
Bias (%)
重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4
Kefeng 6
5.0		 SPS 175,80.0 180,00.0 183,20.0 191,00.0 182,50.0
Kefeng 6 105,8.0 103,2.0 102,7.0 114,2.0 106,4.8 5.83 5.49 16.60
GBH119
1.0		 Adhc 109,000.0 114,000.0 119,400.0 113,800.0 114,050.0
GHB119 952.0 946.0 984.0 916.0 949.5 0.83 3.47 -17.00

Table 3

Quantitative results of G6H1 and GBH119 with simplex droplet dPCR"

样品含量
Sample
content (%)		 靶标
Target		 拷贝数Copies (20 μL-1) 平均值
Average		 转基因含量
GM content (%)		 相对标准差RSD (%) 偏差
Bias (%)
重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4
Kefeng 6
5.0		 SPS 175,80.0 180,00.0 183,20.0 191,00.0 182,50.0
Kefeng 6 105,8.0 103,2.0 102,7.0 114,2.0 106,4.8 5.83 5.49 16.60
GBH119
1.0		 Adhc 109,000.0 114,000.0 119,400.0 113,800.0 114,050.0
GHB119 952.0 946.0 984.0 916.0 949.5 0.83 3.47 -17.00

Table 4

Quantitative results of MON810 and GTS40-3-2 with duplex droplet dPCR"

样品含量
Sample
content		 靶标
Target		 拷贝数Copies (20 μL-1) 转基因含量GM content (%) 平均值
Average (%)		 相对标
准差
RSD (%)		 偏差
Bias
(%)
重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4		 重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4
GTS40-3-2
0.1%		 GTS 5.5 4.8 7.4 7.8 0.09 0.08 0.12 0.13 0.11 23.80 10.00
Lectin 626,0.0 602,0.0 602,0.0 600,0.0
GTS40-3-2
0.5%		 GTS 58.0 64.0 76.0 46.0 0.44 0.49 0.60 0.35 0.47 22.57 6.00
Lectin 131,40.0 129,80.0 126,00.0 132,20.0
GTS40-3-2
1.0%		 GTS 110.0 106.0 108.0 108.0 1.12 1.10 1.13 1.13 1.12 1.42 12.00
Lectin 986,0.0 966,0.0 952,0.0 960,0.0
MON810
0.1%		 MON810 6.8 6.8 8.8 10.0 0.08 0.08 0.11 0.13 0.10 21.73 0.00
Adh-1 808,0.0 836,0.0 800,0.0 802,0.0
MON810
0.5%		 MON810 55.0 54.0 62.0 56.0 0.51 0.49 0.57 0.52 0.52 6.14 4.00
Adh-1 108,40.0 109,80.0 109,60.0 108,60.0
MON810
1.0%		 MON810 196.0 202.0 124.0 166.0 1.21 1.24 0.78 1.04 1.07 19.75 7.00
Adh-1 161,80.0 162,80.0 158,80.0 159,40.0

Table 4

Quantitative results of MON810 and GTS40-3-2 with duplex droplet dPCR"

样品含量
Sample
content		 靶标
Target		 拷贝数Copies (20 μL-1) 转基因含量GM content (%) 平均值
Average (%)		 相对标
准差
RSD (%)		 偏差
Bias
(%)
重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4		 重复1
Repeat 1		 重复2
Repeat 2		 重复3
Repeat 3		 重复4
Repeat 4
GTS40-3-2
0.1%		 GTS 5.5 4.8 7.4 7.8 0.09 0.08 0.12 0.13 0.11 23.80 10.00
Lectin 626,0.0 602,0.0 602,0.0 600,0.0
GTS40-3-2
0.5%		 GTS 58.0 64.0 76.0 46.0 0.44 0.49 0.60 0.35 0.47 22.57 6.00
Lectin 131,40.0 129,80.0 126,00.0 132,20.0
GTS40-3-2
1.0%		 GTS 110.0 106.0 108.0 108.0 1.12 1.10 1.13 1.13 1.12 1.42 12.00
Lectin 986,0.0 966,0.0 952,0.0 960,0.0
MON810
0.1%		 MON810 6.8 6.8 8.8 10.0 0.08 0.08 0.11 0.13 0.10 21.73 0.00
Adh-1 808,0.0 836,0.0 800,0.0 802,0.0
MON810
0.5%		 MON810 55.0 54.0 62.0 56.0 0.51 0.49 0.57 0.52 0.52 6.14 4.00
Adh-1 108,40.0 109,80.0 109,60.0 108,60.0
MON810
1.0%		 MON810 196.0 202.0 124.0 166.0 1.21 1.24 0.78 1.04 1.07 19.75 7.00
Adh-1 161,80.0 162,80.0 158,80.0 159,40.0

Fig. 4

Qualitative PCR detection of transgenic components in S7 and S8 samples stored for six years M: 100 bp DNA ladder; 1-3: sample S7; 4-6: sample S8; 7-9: positive control with GM content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: extract blank control; 16-18: blank H2O control; S7 and S8 are the capability verification samples retained in 2014 with a shelf life of six years."

Fig. 4

Qualitative PCR detection of transgenic components in S7 and S8 samples stored for six years M: 100 bp DNA ladder; 1-3: sample S7; 4-6: sample S8; 7-9: positive control with GM content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: extract blank control; 16-18: blank H2O control; S7 and S8 are the capability verification samples retained in 2014 with a shelf life of six years."

Fig. 5

Qualitative PCR detection of transgenic components in S9 and S10 samples stored for 11 year M: 100 bp DNA ladder; 1-3: sample S9; 4-6: sample S10; 7-9: positive control with GM content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: extract blank control; 16-18: PCR blank H2O control; S9 and S10 are the capability verification samples retained in 2009 with a shelf life of 11 years."

Fig. 5

Qualitative PCR detection of transgenic components in S9 and S10 samples stored for 11 year M: 100 bp DNA ladder; 1-3: sample S9; 4-6: sample S10; 7-9: positive control with GM content 1.0% (w/w); 10-12: non-transgenic negative control; 13-15: extract blank control; 16-18: PCR blank H2O control; S9 and S10 are the capability verification samples retained in 2009 with a shelf life of 11 years."

[1] 于亚东, 卢晓华, 倪晓丽, 陈文祥, 胡晓燕, 吴忠祥, 王亚平. 标准物质通用术语和定义. JJF 1005-2016. 北京: 中国标准出版社, 2016. pp 1-22.
Yu Y D, Lu X H, Ni X L, Chen W X, Hu X Y, Wu Z X, Wang Y P. General Terms and Definitions Used in Connection with Reference Materials. JJF 1005-2016. Beijing: Standards Press of China, 2016. pp 1-22(in Chinese).
[2] 沈平, 李昂, 章秋艳, 李文龙, 宋贵文. 转基因产品标准物质概述. 中国生物工程杂志, 2015, 35:107-111.
Shen P, Li A, Zhang Q Y, Li W L, Song G W. Overview of reference materials for genetically modified products. China Biotechnol, 2015, 35:107-111 (in Chinese with English abstract).
[3] Galvan E C, Torres M, Urquiza M P. Zea maize reference materials for genetically modified organism detection in Mexico. Ecol Evol, 2019, 9:12353-12356.
[4] 刘信. 适于转基因产品检测的核酸扩增技术. 中国农业科技导报, 2011, 13(6):78-81.
Liu X. Alternative DNA amplification method propitious to GMO detection. J Agric Sci Technol, 2011, 13(6):78-81 (in Chinese with English abstract).
[5] 李亮, 王晶, 隋志伟, 臧超, 余笑波. 转基因定量检测用质粒分子标准物质研究进展. 生物技术通报, 2012, (2):54-58.
Li L, Wang J, Sui Z W, Zang C, Yu X B. Advances in reference plasmids for the quantitative detection of genetically modified crops. Biotechnol Bull, 2012, (2):54-58 (in Chinese with English abstract).
[6] Corbisier P, Trapmann S, Gancberg D, Hannes L, Iwarrrden P V, Berben G, Schimmel H, Emons H. Quantitative determination of Roundup Ready soybean (Glycine max) extracted from highly processed flour. Anal Bioanal Chem, 2005, 383:282-290.
[7] 董莲华, 赵正宜, 李亮, 隋志伟, 王晶. 转基因植物标准物质研究进展. 农业生物技术学报, 2012, 20:203-210.
Dong L H, Zhao Z Y, Li L, Sui Z W, Wang J. Advance in developing genetically modified plant reference material. J Agric Biotechnol, 2012, 20:203-210 (in Chinese with English abstract).
[8] 王颢潜, 李夏莹, 张丽, 赵新, 陈锐, 陈笑芸, 高芳瑞, 兰青阔, 王永, 张秀杰. 转基因产品检测标准物质量值一致性研究进展. 生物技术通报, 2020, 36(5):2-8.
Wang H Q, Li X Y, Zhang L, Zhao X, Chen R, Chen X Y, Gao F R, Lan Q K, Wang Y, Zhang X J. Research progress on the trait value consistency of reference materials for genetically modified organism. Biotechnol Bull, 2020, 36(5):2-8 (in Chinese with English abstract).
[9] 李俊, 李夏莹, 李亮, 宋贵文, 沈平, 张丽, 翟杉杉, 柳方方, 吴刚, 张秀杰, 武玉花. 转基因大豆MON89788纯品粉末标准物质的研制及定值. 农业生物技术学报, 2020, 28:1084-1095.
Li J, Li X Y, Li L, Song G W, Shen P, Zhang L, Zhai S S, Liu F F, Wu G, Zhang X J, Wu Y H. Development and characterization of pure matrix reference materials for detection of transgenic soybean (Glycine max) MON89788. J Agric Biotechnol, 2020, 28:1084-1095 (in Chinese with English abstract).
[10] 董莲华, 隋志伟, 沈平, 余笑波, 王晶. 转基因玉米NK603基体标准物质研制. 农业生物技术学报, 2013, 21:12-18.
Dong L H, Sui Z W, Shen P, Yu X B, Wang J. Development of genetically modified maize (Zea mays) line NK603 matrix reference material. J Agric Biotechnol, 2013, 21:12-18 (in Chinese with English abstract).
[11] 李俊, 李亮, 李夏莹, 宋贵文, 沈平, 张丽, 翟杉杉, 柳方方, 吴刚, 张秀杰, 武玉花. 转基因玉米MIR604基体标准物质研制. 作物学报, 2020, 46:473-483.
Li J, Li L, Li X Y, Song G W, Shen P, Zhang L, Zhai S S, Liu F F, Wu G, Zhang X J, Wu Y H. Development of genetically modified maize MIR604 matrix reference materials. Acta Agron Sin, 2020, 46:473-483 (in Chinese with English abstract).
[12] Bainard L D, Klironomos J N, Hart M M. Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA. J Microbiol Methods, 2010, 82:124-130.
[13] 梁子英, 刘芳. 实时荧光定量PCR技术及其应用研究进展. 现代农业科技, 2020, (6):1-3.
Liang Z Y, Liu F. Research progress on real-time quantitative PCR technology and its application. Mod Agric Sci Technol, 2020, (6):1-3 (in Chinese).
[14] 张宜文, 赵海波, 吴红, 马康. 数字PCR在食品安全检测中的应用研究进展. 分析测试学报, 2020, 39:672-680.
Zhang Y W, Zhao H B, Wu H, Ma K. Progress on application of digital PCR in food safety detection. J Instrum Anal, 2020, 39:672-680 (in Chinese with English abstract).
[15] 谢家建, 沈平, 彭于发, 李葱葱, 宋贵文, 孙爻. 转基因植物及其产品成分检测: 调控元件CaMV 35S启动子、FMV 35S启动子、NOS启动子、NOS终止子和CaMV 35S终止子定性PCR方法. 农业部1782号公告-3-2012. 北京: 中国农业出版社, 2012. pp 1-9.
Xie J J, Shen P, Peng Y F, Li C C, Song G W, Sun Y. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Methods for the Regulatory Elements CaMV 35S Promoter, FMV 35S Promoter, NOS Promoter, NOS Terminator and CaMV 35S Terminator. Announcement No. 1782 of the Ministry of Agriculture-3-2012. Beijing: China Agriculture Press, 2012. pp 1-9(in Chinese).
[16] 路兴波, 宋贵文, 李凡, 沈平, 杨立桃, 孙红炜, 武海斌, 王敏, 王鹏. 转基因植物及其产品成分检测: bar或pat基因定性PCR方法. 农业部1782号公告-6-2012. 北京: 中国农业出版社, 2012. pp 1-5.
Lu X B, Song G W, Shen P, Yang L T, Sun H W, Wu H B, Wang M, Wang P. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method of bar or pat Gene. Announcement No. 1782 of the Ministry of Agriculture-6-2012. Beijing: China Agriculture Press, 2012. pp 1-5(in Chinese).
[17] 卢长明, 宋贵文, 吴刚, 武玉花, 曹应龙, 厉建萌, 罗军玲. 转基因植物及其产品成分检测:标记基因NPTII、HPT和PMI定性PCR方法. 农业部1782号公告-2-2012. 北京: 中国农业出版社, 2012. pp 1-9.
Lu C M, Song G W, Wu G, Wu Y H, Cao Y L, Li J M, Luo J L. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Methods for the Marker Genes NPTII, HPT and PMI. No. Announcement No. 1782 of the Ministry of Agriculture -2-2012. Beijing: China Agriculture Press, 2012. pp 1-9(in Chinese).
[18] 金芜军, 刘信, 杨立桃, 张永军, 黄昆龙, 宋贵文, 李宁, 沈平, 彭于发, 黄文胜, 宛煜嵩. 转基因植物及其产品成分检测: 抗虫转Bt基因水稻定性PCR方法. 农业部953号公告-6-2007. 北京: 中国农业出版社, 2007. pp 1-8.
Jin W J, Liu X, Yang L T, Zhang Y J, Huang K L, Song G W, L I N, Shen P, Peng Y F, Huang W S, Wan Y S. Detection of Genetically Modified Plants and Their Derived Products: Qualitative PCR Methods for Pest-Resistant Rice Transgenic for Bt Gene. No. Announcement No. 953 of the Ministry of Agriculture-6-2007. Beijing: China Agriculture Press, 2007. pp 1-8(in Chinese).
[19] 杨立桃, 厉建萌, 刘勇, 张大兵, 宋贵文, 兰青阔, 郭金超, 宋君. 转基因植物及其产品成分检测:CP4-epsps基因定性PCR方法. 农业部1861号公告-5-2012. 北京: 中国农业出版社, 2012. pp 1-5.
Yang L T, Li J M, Liu Y, Zhang D B, Song G W, Lan Q K, Guo J C, Song J. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for CP4-epsps Gene. Announcement No. 1861 of the Ministry of Agriculture-5-2012. Beijing: China Agriculture Press, 2012. pp 1-5(in Chinese).
[20] 杨崇良, 沈平, 路兴波, 孙红炜, 金芜军, 尚佑芬, 赵玖华, 宋贵文, 李宁. 转基因植物及其产品成分检测: 耐除草剂玉米NK603及其衍生品种定性PCR方法. 农业部869号公告-13-2007. 北京: 中国农业出版社, 2007. pp 1-4.
Yang C L, Shen P, Lu X B, Sun H W, Jin W J, Shang Y F, Zhao J H, Song G W, Li N. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Herbicide- tolerant Maize NK603 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture-13-2007. Beijing: China Agriculture Press, 2007. pp 1-4(in Chinese).
[21] 卢长明, 沈平, 武玉花, 黄文胜, 金芜军, 祝长青. 转基因植物及其产品成分检测: 抗虫水稻TT51-1及其衍生品种定性PCR方法. 农业部1193号公告-3-2009. 北京: 中国农业出版社, 2009. pp 1-4.
Lu C M, Shen P, Wu Y H, Huang W S, Jin W J, Zhu C Q. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-Resistant Rice TT51-1 and Its Derivates. Announcement No. 1193 of the Ministry of Agriculture-3-2009. Beijing: China Agriculture Press, 2009. pp 1-4(in Chinese).
[22] 张明, 厉建萌, 潘良文, 杨立桃, 李飞武, 刘乐庭, 刘信, 潘爱虎, 付仲文, 李葱葱. 转基因植物及其产品成分检测:抗除草剂油菜GT73及其衍生品种定性PCR方法.农业部869号公告-11-2007. 北京: 中国农业出版社, 2007. pp 1-4.
Zhang M, Li J M, Pan L W, Yang L T, Li F W, Liu L T, Liu X, Pan A H, Fu Z W, Li C C. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Herbicide-tolerant Canola GT73 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture -11-2007. Beijing: China Agriculture Press, 2007. pp 1-4(in Chinese).
[23] 卢长明, 武玉花, 宋贵文, 吴刚, 肖玲, 沈平, 连庆. 转基因植物及其产品成分检测: 抗除草剂油菜MS8、RF3及其衍生品种定性PCR方法. 农业部869号公告-5-2007. 北京: 中国农业出版社, 2007. pp 1-5.
Lu C M, Wu Y H, Song G W, Wu G, Xiao L, Shen P, Lian Q. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Herbicide-Tolerant Rapeseed MS8, PF3 and Their Derivates. Announcement No. 869 of the Ministry of Agriculture-5-2007. Beijing: China Agriculture Press, 2007. pp 1-5(in Chinese).
[24] 李飞武, 宋贵文, 夏蔚, 沈平, 李葱葱, 李昂, 董立明, 龙立坤, 闫伟, 张明, 邵改革, 徐俊峰, 陈笑芸. 转基因植物及其产品成分检测: 抗虫和耐除草剂玉米Bt11及其衍生品种定性PCR方法. 农业部2122号公告-14-2014. 北京: 中国农业出版社, 2014. pp 1-5.
Li F W, Song G W, Xia W, Shen P, Li C C, Li A, Dong L M, Long L K, Yan W, Zhang M, Shao G G, Xu J F, Chen X Y. Detection of Genetically Modified Plants and Derived Products:Qualitative PCR Method for Insect-Resistant and Herbicide-Tolerant Maize Bt11 and Its Derivates. Announcement No. 2122 of the Ministry of Agriculture-14-2014. Beijing: China Agriculture Press, 2014. pp 1-5(in Chinese).
[25] 易小平, 李文龙, 郭安平, 沈平, 李美英, 李昂, 肖苏生, 贺萍萍, 杨小亮, 谢翔. 转基因植物及其产品成分检测: 抗虫玉米MON810及其衍生品种定性PCR方法. 农业部2122号公告-16-2014. 北京: 中国农业出版社, 2014. pp 1-5.
Yi Y P, Li W L, G uo A P, Shen P, Li M Y, Li A, Xiao S S, He P P, Yang X L, Xie X. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-Resistant Maize MON810 and Its Derivates. Announcement No. 2122 of the Ministry of Agriculture-16-2014. Beijing: China Agriculture Press, 2014. pp 1-5(in Chinese).
[26] 刘勇, 沈平, 宋君, 张瑞英, 雷绍荣, 金芜军, 赵欣, 张富丽, 尹全, 王东, 刘文娟, 常丽娟, 关海涛, 王伟威. 转基因植物及其产品成分检测:耐除草剂大豆GTS40-3-2及其衍生品种定性PCR方法. 农业部1861号公告-2-2012. 北京: 中国农业出版社, 2012. pp 1-5.
刘勇, 沈平, 宋君, 张瑞英, 雷绍荣, 金芜军, 赵欣, 张富丽, 尹全, 王东, 刘文娟, 常丽娟, 关海涛, 王伟威. 转基因植物及其产品成分检测:耐除草剂大豆GTS40-3-2及其衍生品种定性PCR方法. 农业部1861号公告-2-2012. 北京: 中国农业出版社, 2012. pp 1-5.
Liu Y, Shen P, Song J, Zhang R Y, Lei S R, Jin W J, Zhao X, Zhang F L, Yin Q, Wang D, Liu W J, Chang L J, Guan H T, Wan W W. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Herbicide-Tolerant Soybean GTS40-3-2 and Its Derivates. Announcement No. 1861 of the Ministry of Agriculture-2-2012. Beijing: China Agriculture Press, 2012. pp 1-5(in Chinese).
Liu Y, Shen P, Song J, Zhang R Y, Lei S R, Jin W J, Zhao X, Zhang F L, Yin Q, Wang D, Liu W J, Chang L J, Guan H T, Wan W W. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Herbicide-Tolerant Soybean GTS40-3-2 and Its Derivates. Announcement No. 1861 of the Ministry of Agriculture-2-2012. Beijing: China Agriculture Press, 2012. pp 1-5(in Chinese).
[27] 张大兵, 刘信, 杨立桃, 潘爱虎, 沈平, 宋贵文, 路兴波. 转基因植物及其产品成分检测:抗虫玉米MON863及其衍生品种定性PCR方法. 农业部869号公告-10-2007. 北京: 中国农业出版社, 2007. pp 1-4.
张大兵, 刘信, 杨立桃, 潘爱虎, 沈平, 宋贵文, 路兴波. 转基因植物及其产品成分检测:抗虫玉米MON863及其衍生品种定性PCR方法. 农业部869号公告-10-2007. 北京: 中国农业出版社, 2007. pp 1-4.
Zhang D B, Liu X, Yang L T, Pan A H, Shen P, Song G W, Lu X B. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-resistant Maize MON863 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture-10-2007. Beijing: China Agriculture Press, 2007. pp 1-4(in Chinese).
Zhang D B, Liu X, Yang L T, Pan A H, Shen P, Song G W, Lu X B. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-resistant Maize MON863 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture-10-2007. Beijing: China Agriculture Press, 2007. pp 1-4(in Chinese).
[28] 张瑞英, 李文龙, 温洪涛, 沈平, 关海涛, 王伟威, 丁一佳, 黄盈莹. 转基因植物及其产品成分检测:抗虫和耐除草剂玉米Bt176及其衍生品种定性PCR方法. 农业部2122号公告-15-2014. 北京: 中国农业出版社, 2014. pp 1-5.
张瑞英, 李文龙, 温洪涛, 沈平, 关海涛, 王伟威, 丁一佳, 黄盈莹. 转基因植物及其产品成分检测:抗虫和耐除草剂玉米Bt176及其衍生品种定性PCR方法. 农业部2122号公告-15-2014. 北京: 中国农业出版社, 2014. pp 1-5.
Zhang R Y, Li W L, Wen H T, Shen P, Guan H T, Wang W W, Ding Y J, Huang Y Y. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect- Resistant and Herbicide-Tolerance Maize Bt176 and Its Derivates. Announcement No. 2122 of the Ministry of Agriculture-15-2014. Beijing: China Agriculture Press, 2014. pp 1-5(in Chinese).
Zhang R Y, Li W L, Wen H T, Shen P, Guan H T, Wang W W, Ding Y J, Huang Y Y. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect- Resistant and Herbicide-Tolerance Maize Bt176 and Its Derivates. Announcement No. 2122 of the Ministry of Agriculture-15-2014. Beijing: China Agriculture Press, 2014. pp 1-5(in Chinese).
[29] 张大兵, 刘信, 杨立桃, 宋贵文, 沈平, 潘爱虎, 梁婉琪, 张明. 转基因植物及其产品成分检测: 抗虫和耐除草剂玉米TC1507及其生品种定性PCR方法. 农业部869号公告-7-2007. 北京: 中国农业出版社, 2007. pp 1-4.
张大兵, 刘信, 杨立桃, 宋贵文, 沈平, 潘爱虎, 梁婉琪, 张明. 转基因植物及其产品成分检测: 抗虫和耐除草剂玉米TC1507及其生品种定性PCR方法. 农业部869号公告-7-2007. 北京: 中国农业出版社, 2007. pp 1-4.
Zhang D B, Liu X, Yang L T, Song G W, Shen P, Pan A H, Liang W Q, Zhang M. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-Resistant and Herbicide-Tolerant Maize TC1507 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture-7-2007. Beijing: China Agriculture Press, 2014. pp 1-4(in Chinese).
Zhang D B, Liu X, Yang L T, Song G W, Shen P, Pan A H, Liang W Q, Zhang M. Detection of Genetically Modified Plants and Derived Products: Qualitative PCR Method for Insect-Resistant and Herbicide-Tolerant Maize TC1507 and Its Derivates. Announcement No. 869 of the Ministry of Agriculture-7-2007. Beijing: China Agriculture Press, 2014. pp 1-4(in Chinese).
[30] 彭于发, 沈平, 谢家建, 张永军, 厉建萌. 转基因植物及其产品成分检测: 抗虫耐除草剂玉米59122及其衍生品种定性PCR方法. 农业部1485号公告-9-2010. 北京: 中国农业出版社, 2010. pp 1-5.
彭于发, 沈平, 谢家建, 张永军, 厉建萌. 转基因植物及其产品成分检测: 抗虫耐除草剂玉米59122及其衍生品种定性PCR方法. 农业部1485号公告-9-2010. 北京: 中国农业出版社, 2010. pp 1-5.
Peng Y F, Shen P, Xie J J, Zhang Y J, Li J M. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant and herbicide-tolerant maize 59122 and its derivates. Announcement No. 1485 of the Ministry of Agriculture-9-2010. Beijing: China Agriculture Press, 2010. pp 1-5(in Chinese).
Peng Y F, Shen P, Xie J J, Zhang Y J, Li J M. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant and herbicide-tolerant maize 59122 and its derivates. Announcement No. 1485 of the Ministry of Agriculture-9-2010. Beijing: China Agriculture Press, 2010. pp 1-5(in Chinese).
[31] 卢长明, 刘信, 武玉花, 吴刚, 岳云峰, 曹应龙, 赵欣. 转基因植物及其产品成分检测:抗虫玉米MIR604及其衍生品种定性PCR方法. 农业部1485号公告-16-2010. 北京: 中国农业出版社, 2010. pp 1-5.
卢长明, 刘信, 武玉花, 吴刚, 岳云峰, 曹应龙, 赵欣. 转基因植物及其产品成分检测:抗虫玉米MIR604及其衍生品种定性PCR方法. 农业部1485号公告-16-2010. 北京: 中国农业出版社, 2010. pp 1-5.
Lu C M, Liu X, Wu Y H, Wu G, Yue Y F, Cao Y L, Zhao X. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant maize MIR604 and its derivates. Announcement No. 1485 of the Ministry of Agriculture-16-2010. Beijing: China Agriculture Press, 2010. pp 1-5(in Chinese).
Lu C M, Liu X, Wu Y H, Wu G, Yue Y F, Cao Y L, Zhao X. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant maize MIR604 and its derivates. Announcement No. 1485 of the Ministry of Agriculture-16-2010. Beijing: China Agriculture Press, 2010. pp 1-5(in Chinese).
[32] 谢家建, 沈平, 彭于发, 梁晋刚, 陈秀萍, 韩兰芝, 李云河, 梁革梅, 王叶. 转基因植物及其产品成分检测: 抗虫耐除草剂棉花GHB119及其衍生品种定性PCR方法. 农业农村部公告第111号-3-2018. 北京: 中国农业出版社, 2018. pp 1-7.
谢家建, 沈平, 彭于发, 梁晋刚, 陈秀萍, 韩兰芝, 李云河, 梁革梅, 王叶. 转基因植物及其产品成分检测: 抗虫耐除草剂棉花GHB119及其衍生品种定性PCR方法. 农业农村部公告第111号-3-2018. 北京: 中国农业出版社, 2018. pp 1-7.
Xie J J, Shen P, Peng Y F, Liang J G, Chen X P, Han L Z, Li Y H, Liang G M, Wang Y. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant and herbicide-tolerant cotton GHB119 and its derivates. Announcement No. 111 of the Ministry of Agriculture and Rural Affairs -3-2018. Beijing: China Agriculture Press, 2018. pp 1-7(in Chinese).
Xie J J, Shen P, Peng Y F, Liang J G, Chen X P, Han L Z, Li Y H, Liang G M, Wang Y. Detection of genetically modified plants and derived products:Qualitative PCR method for insect-resistant and herbicide-tolerant cotton GHB119 and its derivates. Announcement No. 111 of the Ministry of Agriculture and Rural Affairs -3-2018. Beijing: China Agriculture Press, 2018. pp 1-7(in Chinese).
[33] 徐俊峰, 沈平, 陈笑芸, 宋贵文, 汪小福, 吴刚, 刘慧, 武玉花, 马卉, 朱姝晴, 缪青梅, 赵紫斌, 李昂, 秦瑞英. 转基因植物及其产品成分检测: 抗虫耐除草剂水稻G6H1及其衍生品种定性PCR方法. 农业部2259号公告-11-2015. 北京: 中国农业出版社, 2015. pp 1-5.
徐俊峰, 沈平, 陈笑芸, 宋贵文, 汪小福, 吴刚, 刘慧, 武玉花, 马卉, 朱姝晴, 缪青梅, 赵紫斌, 李昂, 秦瑞英. 转基因植物及其产品成分检测: 抗虫耐除草剂水稻G6H1及其衍生品种定性PCR方法. 农业部2259号公告-11-2015. 北京: 中国农业出版社, 2015. pp 1-5.
Xu J F, Shen P, Chen X Y, Song G W, Wang X F, Wu G, Liu H, Wu Y H, Ma H, Zhu S Q, Miao Q M, Zhao Z B, Li A, Qin R Y. Detection of genetically modified plants and derived products: Qualitative PCR method for insect-resistant and herbicide- tolerant rice G6H1 and its derivates. Announcement No. 2259 of the Ministry of Agriculture-11-2015. Beijing: China Agriculture Press, 2015. pp 1-5(in Chinese).
Xu J F, Shen P, Chen X Y, Song G W, Wang X F, Wu G, Liu H, Wu Y H, Ma H, Zhu S Q, Miao Q M, Zhao Z B, Li A, Qin R Y. Detection of genetically modified plants and derived products: Qualitative PCR method for insect-resistant and herbicide- tolerant rice G6H1 and its derivates. Announcement No. 2259 of the Ministry of Agriculture-11-2015. Beijing: China Agriculture Press, 2015. pp 1-5(in Chinese).
[34] 缪青梅, 汪小福, 陈笑芸, 彭城, 徐晓丽, 魏巍, 徐俊锋. 基于双重微滴数字PCR精准定量转基因水稻G6H1的方法研究. 农业生物技术学报, 2019, 27:159-169.
缪青梅, 汪小福, 陈笑芸, 彭城, 徐晓丽, 魏巍, 徐俊锋. 基于双重微滴数字PCR精准定量转基因水稻G6H1的方法研究. 农业生物技术学报, 2019, 27:159-169.
Miao Q M, Wang X F, Chen X Y, Peng C, Xu X L, Wei W, Xu J F. Studies on accurate quantification of genetically modified rice (Oryza sativa) G6H1 based on duplex droplet digital PCR. J Agric Biotechnol, 2019, 27:159-169 (in Chinese with English abstract).
Miao Q M, Wang X F, Chen X Y, Peng C, Xu X L, Wei W, Xu J F. Studies on accurate quantification of genetically modified rice (Oryza sativa) G6H1 based on duplex droplet digital PCR. J Agric Biotechnol, 2019, 27:159-169 (in Chinese with English abstract).
[35] 黄新, 高宏伟, 李想, 凌杏园, 朱水芳, 陈洪俊, 潘良文, 曹际娟, 章桂明. 转基因产品检测: 实时荧光定性聚合酶链式反应PCR检测方法. GB/T 19495.4-2018. 北京: 中国标准出版社, 2018. pp 1-18.
黄新, 高宏伟, 李想, 凌杏园, 朱水芳, 陈洪俊, 潘良文, 曹际娟, 章桂明. 转基因产品检测: 实时荧光定性聚合酶链式反应PCR检测方法. GB/T 19495.4-2018. 北京: 中国标准出版社, 2018. pp 1-18.
Huang X, Gao H W, Li X, Ling X Y, Zhu S F, Chen H J, Pan L W, Cao J J, Zhang G M. Detection of Genetically Modified Organisms and Derived Products-Qualitative Real-time Polymerase Chain Reaction (PCR) Method. GB/T19495.4-2018. Beijing: China Standards Press, 2018. pp 1-18(in Chinese).
Huang X, Gao H W, Li X, Ling X Y, Zhu S F, Chen H J, Pan L W, Cao J J, Zhang G M. Detection of Genetically Modified Organisms and Derived Products-Qualitative Real-time Polymerase Chain Reaction (PCR) Method. GB/T19495.4-2018. Beijing: China Standards Press, 2018. pp 1-18(in Chinese).
[36] 黄文胜, 陈颖, 邓婷婷, 吴亚君, 付伟, 聂丹丹, 朱水芳, 韩建勋, 李想. 转基因大米定量检测数字PCR法第3部分: 科丰6号品系. SN/T 4853.3-2017. 北京: 中国标准出版社, 2017. pp 1-7.
黄文胜, 陈颖, 邓婷婷, 吴亚君, 付伟, 聂丹丹, 朱水芳, 韩建勋, 李想. 转基因大米定量检测数字PCR法第3部分: 科丰6号品系. SN/T 4853.3-2017. 北京: 中国标准出版社, 2017. pp 1-7.
Huang W S, Chen Y, Deng T T, Wu Y J, Fu W, Nie D D, Zhu S F, Han J X, Li X. Quantitative detection of genetically modified rice-digital PCR-Part 3: Event KF6. SN/T 4853.3-2017. Beijing: China Standards Press, 2015. pp 1-7(in Chinese).
Huang W S, Chen Y, Deng T T, Wu Y J, Fu W, Nie D D, Zhu S F, Han J X, Li X. Quantitative detection of genetically modified rice-digital PCR-Part 3: Event KF6. SN/T 4853.3-2017. Beijing: China Standards Press, 2015. pp 1-7(in Chinese).
[37] 梁文, 付伟, 李亮, 刘刚, 李妍, 车团结, 周李华, 林华, 杨镇州, 朱鹏宇, 马丽侠, 樊春海, 王丽华, 张莹. 转基因植物品系定量检测数字PCR法. GB/T 38132-2019. 北京: 中国标准出版社, 2019. pp 1-7.
梁文, 付伟, 李亮, 刘刚, 李妍, 车团结, 周李华, 林华, 杨镇州, 朱鹏宇, 马丽侠, 樊春海, 王丽华, 张莹. 转基因植物品系定量检测数字PCR法. GB/T 38132-2019. 北京: 中国标准出版社, 2019. pp 1-7.
Liang W, Fu W, Li L, Liu G, Li Y, Che T J, Zhou L H, Lin H, Yang Z Z, Zhu P Y, Ma L X, Fan C H, Wang L H, Zhang Y. Quantitative Determination of Genetically Modified Plants by Digital PCR Method. GB/T 38132-2019. Beijing: China Standards Press, 2019. pp 1-7(in Chinese).
Liang W, Fu W, Li L, Liu G, Li Y, Che T J, Zhou L H, Lin H, Yang Z Z, Zhu P Y, Ma L X, Fan C H, Wang L H, Zhang Y. Quantitative Determination of Genetically Modified Plants by Digital PCR Method. GB/T 38132-2019. Beijing: China Standards Press, 2019. pp 1-7(in Chinese).
[38] Morisset D, Stebih D, Milavec M, Gruden K, Zel J. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One, 2013, 8:e62583.
Morisset D, Stebih D, Milavec M, Gruden K, Zel J. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One, 2013, 8:e62583.
[39] Baoutina A, Bhat S, Partis L, Emslie K R. Storage stability of solutions of DNA standards. Anal Chem, 2019, 91:12268-12274.
Baoutina A, Bhat S, Partis L, Emslie K R. Storage stability of solutions of DNA standards. Anal Chem, 2019, 91:12268-12274.
[40] Guo Y, Yang G Q, Chen Y M, Li D Z, Guo Z H. A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing. Plant Diver, 2018, 40:106-116.
Guo Y, Yang G Q, Chen Y M, Li D Z, Guo Z H. A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing. Plant Diver, 2018, 40:106-116.
[41] Gryson N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem, 2010, 396:2003-2022.
Gryson N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem, 2010, 396:2003-2022.
[42] Anklam E, Gadani F, Heinze P, Pijnenburg H, Van Den Eede G. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur Food Res Technol, 2002, 214:3-26.
Anklam E, Gadani F, Heinze P, Pijnenburg H, Van Den Eede G. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur Food Res Technol, 2002, 214:3-26.
[43] Matsuoka T, Kuribara H, Akiyama H, Miura H, Goda Y, Kusakabe Y, Isshiki K, Toyoda M, Hino A. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize. J Food Hygien Soc Jpn, 2001, 42:24-32.
Matsuoka T, Kuribara H, Akiyama H, Miura H, Goda Y, Kusakabe Y, Isshiki K, Toyoda M, Hino A. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize. J Food Hygien Soc Jpn, 2001, 42:24-32.
[44] Watanable S, Sawada H, Naito S, Akiyama H, Teshima R, Furui S, Kitta K, Hino A. Simulation of collaborative studies for real-time PCR-based quantitative methods for genetically modified crops. J AOAC Int, 2013, 96:357-368.
Watanable S, Sawada H, Naito S, Akiyama H, Teshima R, Furui S, Kitta K, Hino A. Simulation of collaborative studies for real-time PCR-based quantitative methods for genetically modified crops. J AOAC Int, 2013, 96:357-368.
[1] LI Jun,LI Liang,LI Xia-Ying,SONG Gui-Wen,SHEN Ping,ZHANG Li,ZHAI Shan-Shan,LIU Fang-Fang,WU Gang,ZHANG Xiu-Jie,WU Yu-Hua. Development of genetically modified maize MIR604 matrix reference materials [J]. Acta Agronomica Sinica, 2020, 46(4): 473-483.
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