摘要转基因玉米(Zea mays) Bt11是具有抗虫、耐除草剂的转基因玉米品系,目前缺乏对其进行准确定量的检测方法。微滴数字PCR (droplet digital PCR, ddPCR)是近年来新兴的第三代PCR技术,在转基因产品定量领域具有极大的应用前景。为探索微滴数字PCR在转基因植物成分检测中的应用,解决现行转基因食品标准存在的问题,完善我国转基因食品检测监管体系,本研究基于双重微滴数字PCR平台建立转基因玉米Bt11品系及内源基因定量检测方法。选择玉米内源基因Zein及Bt11品系特异性基因Cry1A(b)作为定量靶标序列,从稳定性、特异性、定量线性范围、定量检测低限及准确性分析等方面综合评价双重微滴数字PCR定量检测转基因玉米Bt11品系的方法。实验结果表明,所建立的方法能够实现转基因玉米Bt11品系特异性序列和内源基因的特异性扩增,该方法稳定性好,单位体系内源和外源基因拷贝数在DNA浓度为0.05~10 ng/μL时呈现良好的线性,相关系数R2为0.999;内源和外源基因的定量低限(limit of quantitation, LOQ)分别为11.14和3.96个拷贝,定量准确度试验结果显示相对标准偏差(relative standard deviation, RSD)在5.68%~10.31%之间,满足小于25%的要求。本研究建立的转基因玉米Bt11品系双重微滴数字PCR定量检测方法特异性强、稳定性好、准确度高、定量范围广,可用于食品中转基因玉米品系成分的定量检测。
Abstract：Maize (Zea mays) Bt11 is a genetically modified (GM) maize strain that has the feature of insect-resistant and herbicide-tolerant. At present, there was no accurate quantitative detection methods for Bt11. Droplet digital PCR (ddPCR) is the third generation of PCR technology emerged in recent years, and has great application prospect in quantitative field of genetically modified products. In order to solve the implementation problems of current genetically modified food standards and improve the inspection and supervision of genetically modified foods in China, the application of ddPCR in the screening detection of genetically modified organisms was established. A quantitative detection method for exogenous and endogenous gene of Bt11 was established based on duplex ddPCR. The maize endogenous gene Zein and the specific gene Cry1A(b) of Bt11 were selected as quantitative target sequences. The method of duplex ddPCR for quantitative detection of GM maize Bt11 was evaluated by stability, specificity, quantitative linear range, quantitative detection low limit and accuracy analysis. The results showed that both Bt11 event-specific sequence and endogenous gene could be amplified specifically with good stability. It had an excellent linearity when the DNA concentration is in the range of 0.05~10 ng/μL, and the correlation coefficient R2 was 0.999. The limit of quantitation (LOQ) of endogenous and exogenous genes were 11.14 and 3.96 copies, respectively. The results of quantitative accuracy test showed that the relative standard deviation (RSD) was between 5.68% and 10.31%, meeting the requirement of less than 25%. In conclusion, the quantitative detection method for exogenous and endogenous genes in genetically modified maize Bt11 was established in this study and had strong specificity, good stability, high accuracy and wide quantitative range, which could be used for quantitative detection of the components of GM maize in food.
1 蔡教英, 姚丽锋, 王小玉, 等. 2018. 基于双重微滴式数字PCR对转基因油菜RF1品系的定量方法[J]. 现代食品科技, 34(6): 282-287, 173.(Cai J Y, Yao L F, Wang X Y, et al. 2018. Studies on quantification of genetically modified rapeseed of RF1 with duplex droplet digital polymerase chain reaction (duplex-ddPCR)[J]. Modern Food Science and Technology, 34(6): 282-287+173.) 2 陈雪娇, 宗凯, 孙娟娟, 等. 2018. 转基因水稻品系‘Bt汕优63’微滴式数字PCR定量检测方法的建立[J]. 中国农学通报, 34(6): 131-136. (Chen X J, Zhong K, Sun J J, et al.2018. Establishment of droplet digital PCR quantitative detection method of transgenic rice strain‘Bt Shanyou 63’[J]. Chinese Agricultural Science Bulletin, 34(6): 131-136.) 3 冯兆民, 舒越龙. 2017. 数字PCR技术及其应用进展[J]. 病毒学报, 33(1): 103-107. (Feng Z M, Shu Y L.2017. An overview of digital PCR[J]. Chinese Journal of Virology, 33(1): 103-107.) 4 国家市场监督管理总局, 中国国家标准化管理委员会. GB/T 19495.5-2018. 转基因产品检测实时荧光定量聚合酶链式反应(PCR)检测方法[S]. 北京: 中国标准出版社, 2018. (State Administration for Market Regulation, Standardization Administration of China. GB/T 19495.5-2018. Detection of genetically modified organisms and derived products-Quantitative real-time polymerase chain reaction (PCR) methods[S]. Beijing: China Standard Press, 2018.) 5 国家质量监督检验检疫总局. SN/T 1196-2012. 转基因成分检测玉米检测方法[S]. 北京: 中国标准出版社, 2012. (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. SN/T 1196-2012. Detection of genetically modified components-Maize test methods[S]. Beijing: China Standard Press, 2012.) 6 国家质量监督检验检疫总局. SN/T 1204-2016. 植物及其加工产品中转基因成分实时荧光定性检验方法[S]. 北京: 中国标准出版社, 2016. (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. SN/T 1204-2016. Protocol of the real-time PCR method for detecting genetically modified plants and their derived products[S]. Beijing: China Standard Press, 2016.) 7 国家质量监督检验检疫总局. SN/T 3691-2013. 转基因玉米PCR-DHPLC检测方法[S]. 北京: 中国标准出版社, 2013. (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. SN/T 3691-2013. PCR-DHPL method for detection of genetically modified maize[S]. Beijing: China Standard Press, 2013.) 8 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.) 9 James C.2019. 2018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, 39(8): 1-6. (James C.2019. Development trend of global biotechnology/genetically modified crops commercialization in 2018[J]. China Biotechnology, 39(8): 1-6.) 10 姜羽, 胡佳莹, 杨立桃. 2014. 农业利用微滴数字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.) 11 刘津, 李婷, 冼钰茵, 等. 2018. 转基因大豆MON89788双重数字PCR通用定量检测方法的建立[J]. 食品科学, 39(4): 312-319. (Liu J, Li T, Xian Y Y, et al.2018. An universal quantitative detection method for genetically modified soybean event MON89788 using duplex digital PCR[J]. Food Science, 39(4): 312-319.) 12 农业部. 2014. 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[S]. 北京: 中国标准出版社. (Ministry of agriculture. 2014. Ministry of agriculture no. 2122 bulletin-14-2014. Detection of genetically modified plants and derived products-Qualitative PCR method for insect-resistant and herbicide-tolerant maize Bt11 and its derivates[S]. Beijing: China Standard Press.) 13 潘广, 程颖慧, 黄新, 等. 2016. 转基因玉米品系GA21拷贝数百分含量与质量百分含量关系研究[J]. 植物检疫, 30(6): 49-54. (Pan G, Cheng Y H, Huang X, et al.2016. Study on the relationship between the copy number percentage and mass percentage in transgenicmaize event GA21[J]. Plant Quarantine, 30(6): 49-54.) 14 覃文, 曹际娟, 朱水芳. 2003. 加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测[J]. 生物技术通报, (6): 46-50. (Qin W, Cao J J, Zhu S F. 2003. Quantitative/Identified detection of the genetically modified maize Bt11 components in processed products[J]. Biotechnology Bulletin, (6): 46-50.) 15 任怡菲, 高琴, 邓婷婷, 等. 2016. 基于数字PCR的转基因水稻LL62品系精准定量检测方法建立[J]. 生物技术通报, 32(8): 69-76. (Ren Y F, Gao Q, Deng T T, et al.2016. Establishment of precisely quantitative method of genetically modified rice LL62 based on digital PCR[J]. Biotechnology Bulletin, 32(8): 69-76.) 16 于晓帆, 高宏伟, 孙敏, 等. 2016. 荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆[J]. 食品科学, 37(16): 235-241. (Yu X F, Gao H W, Sun M, et al.2016. Detection of genetically modified soybean event DAS-44406-6 by real-time PCR method and digital PCR method[J]. Food Science, 37(16): 235-241.) 17 张佳玲, 潘广, 章桂明, 等. 2017. 微滴式数字PCR定量检测转基因玉米品系VCO-01981-5[J]. 食品科学, 38(12): 246-252. (Zhang J L, Pan G, Zhang G M, et al.2017. Quantitative detection of transgenic maize event VCO-01981-5 with droplet digital PCR[J]. Food Science, 38(12): 246-252.) 18 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. 19 Corbisier P, Bhat S, Partis L, et al.2010. Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction[J]. Analytical and Bioanalytical Chemistry, 396(6): 2143-2150. 20 Dreo T, Pirc M, Ramsak Z, et al.2014. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: A case study of fire blight and potato brown rot[J]. Analytical and Bioanalytical Chemistry, 406(26): 6513-6528. 21 European Network of GMO Laboratories (ENGL). 2015. Definition of minimum performance requirements for analytical methods of GMO testing[EB/OL]. http://gmo-crl.jrc.ec.europa.eu/. 22 Floren C, Wiedemann I, Brenig B, et al.2015. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)[J]. Food Chemistry, 173: 1054-1058. 23 Gasparic M B, Tengs T, Paz J L, et al.2010. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection[J]. Analytical and Bioanalytical Chemistry, 396(6): 2023-2029. 24 Mahboudi H, Heidari N M, Rashidabadi Z I, et al.2018. Prospect and competence of quantitative methods via real-time PCR in a comparative manner: An experimental review of current methods[J]. The Open Bioinformatics Journal, 11(1): 1-11. 25 White R A, Quake S R, Curr K.2012. Digital PCR provides absolute quantitation of viral load for an occult RNA virus[J]. Journal of Virologic Methods, 179(1): 45-50. 26 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.
