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| Establishment of Accurate Quantitative Detection Method for Herbicide-tolerant Soybean (Glycine max) 'DBN9004' |
| ZHAO Xin1, LIU Shuang1, LIU Na1, LI Rui-Huan1, GAO Fang-Rui1, LAN Qing-Kuo1, LIANG Jin-Gang2,*, WANG Yong1,* |
1 Tianjin Academy of Agricultural Sciences, Tianjin 300384, China;
2 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100176, China |
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Abstract Herbicide-tolerant soybean (Glycine) 'DBN9004' is a new genetically modified soybean event with tolerance to both glyphosate and glufosinate. It has obtained safety certificate and has important industrial application prospect in China. In order to meet the technical requirements of safety supervision for genetically modified crops, it is urgent to develop quantitative detection methods for 'DBN9004'. In this study, the 3' flanking sequence of herbicide-tolerant soybean 'DBN9004' was used as the target, primers and probes were designed, and a qPCR assay was established. The specificity, accuracy, precision, detection limit, quantitative limit and other indicators were tested. The results showed that the method was specific and reproducible, with a quantitative limit of 0.1% (40 copies) and a detection limit of 0.05% (20 copies) , the relative standard deviation (RSD) was 6.00%~10.95%, and the quantitative results of qPCR and droplet digital PCR method were consistent. This method provides a new technical means for the accurate quantitative detection of new herbicide-tolerant soybean event 'DBN9004', and provides technical support for the regulation of agricultural transgene crops.
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Received: 13 January 2022
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Corresponding Authors:
* wytaas@126.com; 382408162@qq.com
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[1] 国际农业生物技术应用服务组织. 2021. 2019年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 41(01): 114-119.
(International Service for the Acquisition of Agri-biotech Applications.2021. The development trend of global biotechnology/GM crop commercialization in 2019[J]. Chinese Journal of Biological Engineering, 41(01): 114-119.)
[2] 龙丽坤, 赵宁, 李葱葱, 等. 2021. 转基因玉米CM8101实时荧光定量PCR检测方法的建立[J]. 农业生物技术学报, 29(05): 1007-1015.
(Long L K, Zhao N, Li C C, et al.2021. Establishment of qRT-PCR method of detection transgenic maize (Zea mays) CM8101[J]. Journal of Agricultural Biotechnology, 29(05): 1007-1015.)
[3] 刘双, 赵新, 李瑞环, 等. 2021. 转基因大豆'ZH 10-6'数字PCR精准定量检测方法的建立[J].中国农业大学学报, 26(11): 49-58.
(Liu S, Zhao X, Li R H, et al.2021. Establishment of digital PCR method for quantitative detection of genetically modified soybean 'ZH 10-6'[J]. Journal of China Agricultural University, 26(11): 49-58.)
[4] 雷展, 王建成, 张晨, 等. 2021. 转基因油菜NS-B50027-4定性定量检测方法的建立[J]. 农业生物技术学报, 29(09): 1825-1835.
(Lei Z, Wang J C, Zhang C, et al.2021. Establishment of qualitative and quantitative detection method for transgenic brassica napus NS-B50027-4[J]. Journal of Agricultural Biotechnology,29(09): 1825-1835.)
[5] 斯能武, 李俊, 武玉花, 等. 2021. 数字PCR在转基因定量检测中的研究进展[J]. 中国油料作物学报, 43(01): 40-50.
(Si N W, Li J, Wu Y H, et al.2021. Research progress of digital PCR in quantitative detection of genetically modified organism[J]. Chinese Journal of Oil Crop Sciences, 43(01): 40-50.)
[6] 吴潇, 吕贝贝, 蒋玮, 等. 2018. QX200微滴式数字PCR方法检测转基因大豆GTS-40-3-2[J]. 上海农业学报, 34(01): 14-19.
(Wu X, Lv B B, Jiang W, et al.2018. Development of droplet digital PCR QX200 method for quantifying transgenic soybean GTS-40-3-2[J]. Acta Agriculturae Shanghai, 34(01): 14-19.)
[7] 徐若梅. 2018. 全球转基因作物商业化的发展态势与启示[J]. 安徽农业大学学报(社会科学版), 27(04): 62-67.
(Xu R M.2018. Developing trend and enlightenment of the global commercialization of genetically modified crops[J]. Journal of Anhui Agricultural University (Social Science Edition), 27(04): 62-67.)
[8] 杨晨, 邓嘉慧, 陈佩虹, 等. 2021. 微滴式数字PCR和实时荧光定量PCR检测大豆中的转基因成分[J]. 质量安全与检验检测, 31(05): 14-16.
(Yang C, Deng J H, Chen P H, et al.2021. Detection of genetically modified components in soybean products based on real-time fluorescence quantitative PCR and digital PCR[J]. Quality Safety Inspection and Testing, 31(05): 14-16.)
[9] 赵新, 刘双, 刘娜, 等. 2022. 利用微滴数字PCR技术分析转基因大豆'GE-J12'中外源基因的拷贝数[J]. 中国农业大学学报, 27(01): 58-66.
(Zhao X, Liu S, Liu N, et al.2022. Analysis of the copy number of exogenous gene in transgenic soybean 'GE-J12' with droplet digital PCR[J]. Journal of China Agricultural University, 27(01): 58-66.)
[10] Gong L, Zhang H, Liu X, et al.2020. Ectopic expression of Ha NAC1, an ATAF transcription factor from Haloxylon ammodendron, improves growth and drought tolerance in transgenic Arabidopsis[J]. Plant Physiology and Bio-chemistry, 151: 535-544.
[11] Jiang Y, Yang H, Quan S, et al.2015. Development of certified matrix-based reference material of genetically modified rice event TT51-1 for real-time PCR quantification[J]. Analytical and bioanalytical chemistry, 407(22): 1-9.
[12] Kitta K, Takabatake R, Mano J.2016. Real-time PCR-based quantitation method for the genetically modified soybean line GTS 40-3-2[J]. Methods in Molecular Biology, 1385: 249-257.
[13] Liu J, Li Z Y, Dong J, et al.2020. A universal quantification of transgenic soybean event DAS-68416-4 using duplex digital PCR[J]. Journal of The Science of Food and Agriculture, 101(2): 624-630.
[14] Lou Y Y, Chen C X, Long X L, et al.2020. Detection and quantification of chimeric antigen receptor transgene copy number by droplet digital PCR versus real-time PCR[J]. The Journal of Molecular Diagnostics, 22(5): 699-707.
[15] Wang X F, Tang T, Miao Q M, et al.2018. Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR[J]. Food Control, 98: 135-144.
[16] Zeng H, Wang J, Jia J, et al.2021. Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in geneti-cally modified crops[J]. Food Chemistry, 335(15): 1-6.
[17] Zhuang Y, Yu W X.2013. Improving the enforceability of the genetically modified food labeling law in China with lessons from the European Union[J]. Vermont Journal of Environmental Law, 14(3): 465-492. |
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