利用微滴式数字PCR技术检测大豆毛油中的转基因成分
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摘要
我国是转基因大豆的进口大国,由转基因大豆加工而成的大豆油成为进入消费者生活的食用油,判定食用的大豆油是否含有转基因成分,是国家监管部门和公众消费者密切关心、关注的问题。大豆油在制备精炼过程中破坏了大豆的DNA,造成检验时DNA富集难度增加,严重影响了大豆油转基因成分检测的准确度。到目前为止,国家已经颁布的标准中没有涉及转基因大豆油的检测标准。本研究利用高灵敏度、精确度的数字PCR技术,并通过优化大豆毛油DNA提取方法和PCR反应体系,对大豆毛油样品进行外源转基因成分检测。检测结果显示,大豆毛油外源转基因成分被成功检测到。实验结果表明,本研究建立的大豆毛油转基因成分检测方法准确可靠,可进行推广应用。
China is one of the biggest importers of genetically modified soybeans, and the oil made from the soybeans has become cooking oil for consumers. Whether it contains GM ingredients is a matter of close concern to national regulators and the public. So far, there was no standard method for the detection of GM ingredients in soybean oil, because it was difficult to detect the transgenic components with conventional PCR due to the low concentration and serious degradation of DNA of soybean oil made from genetically modified soybeans. Using the highly sensitive and accurate digital PCR technology, and the optimized extraction method of soybean oil DNA and PCR reaction system in this work, the exogenous transgenic components of soybean oil were successfully detected. The results showed that, an accurate and reliable detection method for the transgenic soybean oil had been established and the proposed method could be applied.
China is one of the biggest importers of genetically modified soybeans, and the oil made from the soybeans has become cooking oil for consumers. Whether it contains GM ingredients is a matter of close concern to national regulators and the public. So far, there was no standard method for the detection of GM ingredients in soybean oil, because it was difficult to detect the transgenic components with conventional PCR due to the low concentration and serious degradation of DNA of soybean oil made from genetically modified soybeans. Using the highly sensitive and accurate digital PCR technology, and the optimized extraction method of soybean oil DNA and PCR reaction system in this work, the exogenous transgenic components of soybean oil were successfully detected. The results showed that, an accurate and reliable detection method for the transgenic soybean oil had been established and the proposed method could be applied.
引文
[1]褚成才.转基因生物育种:机遇还是挑战?[J].植物学报,2013, 48(1):10-22CHU C C. Breeding of genetically modified organisms:Opportunities or challenges?[J]. Journal of Plant, 2013, 48(1):10-22
[2] CLIVE James. 2013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,2014(1):1-8CLIVE James. Global commercialization of biotechnology/gm crops in 2013[J]. Chinese Journal of Bioengineering, 2014(1):1-8
[3]刘定富.中国转基因作物进口的现状和趋势[基因农业网].[2017-06-19]. http://www. agrogene. cn/info-4161. shtml LIU D F. Current situation and trend of import of gm crops in China[Genetic agricultural network].[2017-06-19]. http://www. agrogene. cn/info-4161. shtml
[4]GAWIENOWSKI M C, ECKHOFF SR, YANG P, et al. Fate of maize DNA during steeping, wet-milling, and processing[J]. Cereal Chemistry, 1999, 76(3):371-374
[5] NICOLAS G. Effect of food processing on plant DNA degradation and PCR-based GMO analysis:a review[J]. Anal Bioanal Chem, 2010, 396:2003-2022
[6] SN/T 1195—2003.大豆中转基因成分的定性PCR检测方法[S]SN/T 1195—2003. Qualitative PCR method for detection of transgenic components in soybean[S]
[7] VOGELSTEIN B, KINZLER KW. Digital PCR[J]. Proc Natl Acads Sci. 1999, 96(16):9236-9241
[8]詹成,燕丽,王琳,等.数字PCR技术的发展和应用[J].复旦学报:医学版,2015, 42(6):786-789ZHAN C, YAN L, WANG L, et al. Development and application of digital PCR technology[J]. Journal of fudan university:Medical sciences, 2015, 42(6):786-789
[9] CORBISIER P, BHAT S, PARTIS L, et al. Absolute quantification of genetically modified MON810 maize(Zea mays L.)by digital polymearase chain reaction[J]. Analytical andBioanalytical Chemistry, 2010, 396(6):2143-2150
[10]DANY M, DEJAN T, MOJCA M, et al. Quantitative analysis of food and feed samples with droplet digital PCR[J]PloS One, 2013, 8(5):62583-62583
[11]隋志伟,余笑波,王晶,等.转基因水稻TT51-1标准物质的研制[J].计量学报,2013, 33(5):67-471SUI Z W, YU X B, WANG J, et al. The development of the standard substanceof transgenic rice TT51-1[J]. Journal of measurement, 2013, 33(5):67-471
[12]潘广,章桂明,黄新,等.应用双重数字PCR对转基因玉米成分进行定量方法研究[J].植物检疫,2016, 30(3):65-71PAN G, ZHANG G M, HUANG X, et al. The research of quantitative method of transgenic components in maize using double digital PCR. Plant quarantine, 2016, 30(3):65-71
[13]于晓帆,高宏伟,孙敏,等.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,2016, 37(16):235-241YU X F, GAO H W, SUN M, et al. The detection of DAS-44406-6 transgenic soybean using fluorescent PCR and digital PCR[J]. Food Science, 2016, 37(16):235-241
[14]姜志军,江颖,徐摇光,等.利用微滴数字PCR方法快速分析转基因玉米中外源基因的拷贝数[J].生物技术进展,2016, 6(4):288-294JIANG Z J, JIANG Y, XU Y G, et al. Rapid analysis of the copy number of exogenous genes in genetic maize using microdroplet digital PCR method[J]. Advances in Biotechnology, 2016, 6(4):288-294
[15]付海滨,闫超杰,李姝,等.数字PCR技术在转基因成分检测中的应用[J].辽宁农业科学,2017(1):50-53FU H B, YAN C J, LI S, et al. Application of digital PCR in detection of transgenic components[J]. Liaoning Agricultural Science, 2017(1):50-53
[16]刘津,李婷,冼钰茵,等.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,2018,39(4):312-319LIU J, LI T, XIAN Y Y, et al. Establishment of a universal quantitative detection method for transgenic soybean MON89788 by double digital PCR[J]. Food Science,2018,39(4):312-319
[17] GB/T 33526-2017.转基因植物产品数字PCR检测方法[S]GB/T 33526—2017. Digital PCR assay for transgenic plant products[S].
[2] CLIVE James. 2013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,2014(1):1-8CLIVE James. Global commercialization of biotechnology/gm crops in 2013[J]. Chinese Journal of Bioengineering, 2014(1):1-8
[3]刘定富.中国转基因作物进口的现状和趋势[基因农业网].[2017-06-19]. http://www. agrogene. cn/info-4161. shtml LIU D F. Current situation and trend of import of gm crops in China[Genetic agricultural network].[2017-06-19]. http://www. agrogene. cn/info-4161. shtml
[4]GAWIENOWSKI M C, ECKHOFF SR, YANG P, et al. Fate of maize DNA during steeping, wet-milling, and processing[J]. Cereal Chemistry, 1999, 76(3):371-374
[5] NICOLAS G. Effect of food processing on plant DNA degradation and PCR-based GMO analysis:a review[J]. Anal Bioanal Chem, 2010, 396:2003-2022
[6] SN/T 1195—2003.大豆中转基因成分的定性PCR检测方法[S]SN/T 1195—2003. Qualitative PCR method for detection of transgenic components in soybean[S]
[7] VOGELSTEIN B, KINZLER KW. Digital PCR[J]. Proc Natl Acads Sci. 1999, 96(16):9236-9241
[8]詹成,燕丽,王琳,等.数字PCR技术的发展和应用[J].复旦学报:医学版,2015, 42(6):786-789ZHAN C, YAN L, WANG L, et al. Development and application of digital PCR technology[J]. Journal of fudan university:Medical sciences, 2015, 42(6):786-789
[9] CORBISIER P, BHAT S, PARTIS L, et al. Absolute quantification of genetically modified MON810 maize(Zea mays L.)by digital polymearase chain reaction[J]. Analytical andBioanalytical Chemistry, 2010, 396(6):2143-2150
[10]DANY M, DEJAN T, MOJCA M, et al. Quantitative analysis of food and feed samples with droplet digital PCR[J]PloS One, 2013, 8(5):62583-62583
[11]隋志伟,余笑波,王晶,等.转基因水稻TT51-1标准物质的研制[J].计量学报,2013, 33(5):67-471SUI Z W, YU X B, WANG J, et al. The development of the standard substanceof transgenic rice TT51-1[J]. Journal of measurement, 2013, 33(5):67-471
[12]潘广,章桂明,黄新,等.应用双重数字PCR对转基因玉米成分进行定量方法研究[J].植物检疫,2016, 30(3):65-71PAN G, ZHANG G M, HUANG X, et al. The research of quantitative method of transgenic components in maize using double digital PCR. Plant quarantine, 2016, 30(3):65-71
[13]于晓帆,高宏伟,孙敏,等.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,2016, 37(16):235-241YU X F, GAO H W, SUN M, et al. The detection of DAS-44406-6 transgenic soybean using fluorescent PCR and digital PCR[J]. Food Science, 2016, 37(16):235-241
[14]姜志军,江颖,徐摇光,等.利用微滴数字PCR方法快速分析转基因玉米中外源基因的拷贝数[J].生物技术进展,2016, 6(4):288-294JIANG Z J, JIANG Y, XU Y G, et al. Rapid analysis of the copy number of exogenous genes in genetic maize using microdroplet digital PCR method[J]. Advances in Biotechnology, 2016, 6(4):288-294
[15]付海滨,闫超杰,李姝,等.数字PCR技术在转基因成分检测中的应用[J].辽宁农业科学,2017(1):50-53FU H B, YAN C J, LI S, et al. Application of digital PCR in detection of transgenic components[J]. Liaoning Agricultural Science, 2017(1):50-53
[16]刘津,李婷,冼钰茵,等.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,2018,39(4):312-319LIU J, LI T, XIAN Y Y, et al. Establishment of a universal quantitative detection method for transgenic soybean MON89788 by double digital PCR[J]. Food Science,2018,39(4):312-319
[17] GB/T 33526-2017.转基因植物产品数字PCR检测方法[S]GB/T 33526—2017. Digital PCR assay for transgenic plant products[S].