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| Studies on Accurate Quantification of Genetically Modified Rice (Oryza sativa) G6H1 Based on Duplex Droplet Digital PCR |
| MIAO Qing-Mei, WANG Xiao-Fu*, CHEN Xiao-Yun, PENG Cheng, XU Xiao-Li, WEI Wei, XU Jun-Feng* |
| State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China |
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Abstract Rice (Oryza sativa) G6H1 is a new transgenic rice line that has the feature of insect-resistant and herbicide-tolerant. It is developed by Chinese scientists with independent intellectual property rights. The previous studies showed that G6H1 had good performance of insect and glyphosate resistance, and it met the requirement of application level for insect-resistant and herbicide-tolerant, and had a good and broad application prospect. At present, there was lack of methods for accurate quantitative detection of G6H1. In this study, based on the droplet digital PCR (ddPCR) platform, a duplex ddPCR accurate quantification method for G6H1 was established. First, the specificity of primer and probe designed for the G6H1 event was verified by qRT-PCR. The fluorescent signal was not observed in other transgenic crops and non-transgenic crops and only was observed in G6H1 event, these results indicated that designed primer and probe had high specificity for G6H1; then, the suitable different rice endogenous genes for duplex ddPCR detection method were screened and selected, the sucrose phosphate synthase gene (SPS) with 122 length was chosen as the candidate endogenous gene in this study. Moreover, the concentration and annealing temperature of the primer and probe for duplex ddPCR were optimized. The final concentration of primers/probes as 0.5/0.25 μmol/L combined with 58 ℃ annealing temperature were determined as the final optimal condition for the duplex ddPCR. The established duplex ddPCR method for analysis of transgenic rice G6H1 showed that the limit of detection for endogenous and exogenous genes reached 3 copies, and the limit of quantification of endogenous and exogenous genes was 25 copies, and the relative standard deviation (RSD) of quantitative results was less than 25%. Finally, the quantitative results of samples content of 5%, 1%, and 0.5% of transgenic rice G6H1 were compared using simplex qRT-PCR, simplex ddPCR, and duplex ddPCR. All the results showed that the established duplex ddPCR method had good specificity, high sensitivity, accuracy and reliability, and was suitable for precise quantitative analysis of transgenic rice G6H1 event. The establishment of this method provided an alternative method for quantitative detection of transgenic rice G6H1, and improved the detection technology system for transgenic rice components of China. Meanwhile, the established method also can work as new technical reserves for the safefy threshold regulation of genetically modified agricultural organisms.
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Received: 24 July 2018
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Corresponding Authors:
*, yywxf1981@163.com; njjfxu@163.com
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[1] 陈颖, 黄文胜, 邓婷婷, 等. 2017. SN/T 4853.1-2017. 转基因大米定量检测数字PCR法第1部分: TT51-1品系[S].(Chen Y, Huang W S, Deng T T, et al. 2017. SN/T 4853.1-2017. Quantitative detection of genetically modified rice Digital PCR Part 1: Event TT51-1[S].)
[2] 姜羽, 胡佳莹, 杨立桃. 2014. 利用微滴数字PCR分析转基因生物外源基因拷贝数[J]. 农业生物技术学报, 22(10): 1298-1305.
(Jiang Y, Hu J Y, Yang L T.2014. Estimating the exogenous genes copy number of genetically modified organisms by droplet digital PCR[J]. Journal of Agricultural Biotechnology, 22(10): 1298-1305.)
[3] 金芜军, 沈平, 张秀杰, 等. 2012. 农业部1861号公告- 1-2012. 转基因植物及其产品成分检测-水稻内标准基因定性PCR方法[S]. 中化人民共和国国家标准. (Jin WJ, Shen P, Zhang X J, et al. 2012. Announcement by the Ministry of Agriculture No. 1861-1-2012. Detection of genetically modified plants and derived products-target taxon-specific qualitative PCR method for rice [S]. Nation Standard of the People's Republic of China.)
[4] 胡佳莹, 姜羽, 杨立桃. 2016. 利用QuantStudioTM 3D数字PCR分析转基因玉米MON863含量[J]. 农业生物技术学报, 24(8): 1216-1224.
(Hu J Y, Jiang Y, Yang L T.2016. Quantification of genetically modified maize (Zea mays) MON863 by QuantStudioTM 3D Digital PCR[J]. Journal of Agricultural Biotechnology, 24(8): 1216-1224.)
[5] 李亮, 隋志伟, 王晶, 等. 2012. 基于数字PCR的单分子DNA定量技术研究进展[J]. 生物化学与生物物理进展, 39(10): 1017-1023.
(Li L, Sui Z W, Wang J, et al.2012. Progress of digital PCR for single DNA quantification[J]. Progress in Biochemistry and Biophysics, 39(10): 1017-1023.)
[6] Burns M J, Nixon G J, Foy C A, et al.2005. Standardisation of data from real-time quantitative PCR methods-evaluation of outliers and comparison of calibration curves[J]. BMC Biotechnology, 5(1): 31.
[7] Collier R, Dasgupta K, Xing Y P, et al.2017. Accurate measurement of transgene copy number in crop plants using droplet digital PCR[J]. Plant Journal, 90(5): 1014-1025.
[8] CRL-GMFF.2008. Event-specific method for the quantification of rice line LLRICE62 using real-time PCR protoco (http://gmo-crl.jrc.ec.europa.eu/gmomethods/docs/QT-EVE-OS-002.pdf
[9] Dobnik D, Spilsberg B, Bogozalec K A, et al.2015. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction[J]. Analytical Chemistry, 87(16): 8218-8226.
[10] Demeke T, Holigroski M, Eng M, et al.2016. Absolute quantification of genetically engineered traits with droplet digital PCR: Effect of DNA treatments and spiking with non-target DNA[J]. Food Control, 68: 105-111.
[11] European network of GMO laboratories (ENGL), 2008. Definition of minimum performance requirements for analytical methods of GMO testing. (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf.
[12] Hansulrich W, Britta E, Annette A, et al.2008. Validation and collaborative study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products[J]. European Food Research and Technology, 226(5): 1221-1228.
[13] Lu I J, Lin C H, Pan T M.2010. Establishment of a system based on universal multiplex-PCR for screening genetically modified crops[J]. Analytical and Bioanalytical Chemistry, 396(6): 2055-2064.
[14] Mahboudi H, Heidari N M, Rashidabadi Z I, et al.2018. Prospect and competence of quantitative methods real-time PCR in a comparative manner: An Experimental Review of Current Methods[J]. Open Bioinformatics Journal, 11(1): 1-11.
[15] Morisset D, Stebih D, Milavec M, et al.2013. Quantitative analysis of food and feed samples with droplet digital PCR[J]. PLoS One, 8(5): e62583.
[16] Vogelstein B, Kinzler K W.1999. Digital PCR[J]. Proceedings of the National Academy of Sciences of the USA, 96(16): 9236-9241.
[17] Wang X, Chen X, Xu J, et al.2015. Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes[J]. Food and Chemical Toxicology, 84: 89-98.
[18] Zhao Y, Xia Q, Yin Y, et al.2016. Comparison of droplet digital PCR and quantitative PCR assays for quantitative detection of Xanthomonas citri Subsp.citri[J]. PloS One, 11(7): e0159004. |
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