转基因大豆RPA检测技术的建立及应用
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摘要
旨在建立一种简单可行的转基因大豆RPA检测技术用于国内转基因大豆的监测。根据转基因植物中一般通用的CaMV35S启动子和NOS终止子序列设计了RPA引物,利用常规PCR技术筛选出了一组特异性强且高效的Nos163引物用于试剂条设计,并对RPA反应温度和时间进一步优化建立了转基因大豆的检测体系。该方法建立后,对南京市售的豆芽菜和鲜食豆荚,田间种植的大豆样品进行了检测,没有发现转基因成分的存在。研究结果表明转基因大豆RPA体系具有准确性好,比常规PCR检测技术灵敏度高,操作简便,不需要昂贵仪器,可以常温进行和结果肉眼可见等优点,可用于转基因大豆的快速检测。
The goal of this work is to develop a fast and easy-operating RPA detection method for the GM soybean to monitor the illegal planting and spreading of the GM soybeans in China. Five pairs of RPA primers were initially designed based on the conserved sequences of CaMV-35 S promoter and the NOS terminator in GM soybeans. PCR reactions were further performed to screen the primers and a set of RPA primers with the high sensitivity and specificity,Nos163,was selected for constructing RPA test strips. Reaction temperatures and times of RPA were also optimized. No transgenic component was detected from bean sprouts and fresh green soybean in market,as well as soy samples planted in field in Nanjing area using our RPA detection technique. All evidences suggest that RPA detection method is more sensitive than PCR,and can be performed under normal temperature without depending on the expensive equipment. The test results can be easily visualized,therefore which makes it an effective approach for the rapid detection of GM soybean.
The goal of this work is to develop a fast and easy-operating RPA detection method for the GM soybean to monitor the illegal planting and spreading of the GM soybeans in China. Five pairs of RPA primers were initially designed based on the conserved sequences of CaMV-35 S promoter and the NOS terminator in GM soybeans. PCR reactions were further performed to screen the primers and a set of RPA primers with the high sensitivity and specificity,Nos163,was selected for constructing RPA test strips. Reaction temperatures and times of RPA were also optimized. No transgenic component was detected from bean sprouts and fresh green soybean in market,as well as soy samples planted in field in Nanjing area using our RPA detection technique. All evidences suggest that RPA detection method is more sensitive than PCR,and can be performed under normal temperature without depending on the expensive equipment. The test results can be easily visualized,therefore which makes it an effective approach for the rapid detection of GM soybean.
引文
[1]何超.转基因作物20年[J].湖南农业, 2016, 9:28.
[2]国际农业生物技术应用服务组织. 2017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, 2018, 6:1-8.
[3]沈平,武玉花,梁晋刚,等.转基因作物发展及应用概述[J].中国生物工程杂志, 2017, 37(1):119-128.
[4]杨华,彭城,肖英平,等:转基因大豆SHZD32-1转化体普通PCR和qRT-PCR检测方法的研究[J].农业生物技术学报,2017, 26(3):492-501.
[5]查霆,钟宣伯,周启政,等.我国大豆产业发展现状及振兴策略[J].大豆科学, 2018, 37(3):458-463.
[6]宋扬,吴存祥,侯文胜,等.对引进的美国大豆品种进行转基因成分的检测[J].大豆科学, 2005, 24(2):116-120.
[7]詹琳.全球转基因作物商业化进展情况及有关问题的分析[D].北京:中国农业科学院, 2015.
[8]刘慧.国产大豆产量创十年来新高我国亟需打响“中国大豆”品牌[J].中国食品, 2018, 11:94-95
[9]Singh OV, Ghai S, Paul D, et al. Genetically modified crops:success, safety assessment, and public concern[J]. Appl Microbiol Biotechnol 2006, 71(5):598-607.
[10]崔宁波,张正岩.转基因大豆研究及应用进展[J].西北农业学报, 2016, 25(8):1111-1124.
[11]王宜林,李宏.抗除草剂转基因植物的研究现状[J].生物学通报, 2005, 40(4):15-17.
[12]郭兵福,等.不同大豆基因型对农杆菌敏感性研究及优异种质筛选[J].植物遗传资源学报, 2015, 16(3):569-575.
[13]王志刚,彭纯玉.中国转基因作物的发展现状与展望[J].农业展望, 2010, 6(11):51-55.
[14]沈泓,李超,李珏.分子生物学技术在转基因食品检测领域中的研究进展[J].中国农业信息, 2017, 15:57-59.
[15]Kamle M, et al. Current perspectives on genetically modified crops and detection methods[J]. Biotech, 2017, 7(3):219.
[16]Salisu IB, Shahid AA, Yaqoob A, et al. Molecular approaches for high throughput detection and quantification of genetically modified crops:A review[J]. Front Plant Sci 2017, 8:1670.
[17]Randhawa GJ, et al. Loop-mediated isothermal amplification:rapid visual and real-time methods for detection of genetically modified crops[J]. J Agric Food Chem 2013, 47:11338-11346.
[18]周杰,黄文胜,邓婷婷,等.环介导等温扩增法检测6种转基因大豆[J].农业生物技术学报, 2017, 25(2):335-344.
[19]吕蓓程,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学, 2010, 40(10):983-988.
[20]Piepenburg O, Williams CH, et al. DNA detection using recombination proteins[J]. PLoS Biology, 2006, 4(7):1115-1121.
[21]高威芳,朱鹏,黄海龙.重组酶聚合酶扩增技术:一种新的核酸扩增策略[J].中国生物化学与分子生物学报, 2016, 32(6):627-634.
[22]景志刚,董浩,狄栋栋,等.重组酶聚合酶扩增技术研究进展[J].生物技术通报, 2016, 32(6):47-53.
[23]周延培,孙海新,赵美丽,等.一种转基因马铃薯PCR鉴别方法的建立[J].粮油食品科技, 2018(3):67-70.
[24]Xu C, Li L, Jin W, Wan Y:Recombinase polymerase amplification(RPA)of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops[J]. Int J Mol Sci 2014,15(10):18197-18205.
[25]马建荣,余永红.市场销售大豆的转基因检测[J].广东化工,2018, 45(2):37-38.
[26]钱云开,高飞,王海洋,等. 16种转基因大豆品系的实时荧光PCR法筛查[J].中国油脂, 2017, 42(2):141-145.
[27]Liu M, Luo Y, Tao R, et al. Sensitive and Rapid Detection of Genetic Modified Soybean(Roundup Ready)by Loop-Mediated Isothermal Amplification[J]. Journal of the Agricultural Chemical Society of Japan 2009, 73(11):2365-2369.
[2]国际农业生物技术应用服务组织. 2017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, 2018, 6:1-8.
[3]沈平,武玉花,梁晋刚,等.转基因作物发展及应用概述[J].中国生物工程杂志, 2017, 37(1):119-128.
[4]杨华,彭城,肖英平,等:转基因大豆SHZD32-1转化体普通PCR和qRT-PCR检测方法的研究[J].农业生物技术学报,2017, 26(3):492-501.
[5]查霆,钟宣伯,周启政,等.我国大豆产业发展现状及振兴策略[J].大豆科学, 2018, 37(3):458-463.
[6]宋扬,吴存祥,侯文胜,等.对引进的美国大豆品种进行转基因成分的检测[J].大豆科学, 2005, 24(2):116-120.
[7]詹琳.全球转基因作物商业化进展情况及有关问题的分析[D].北京:中国农业科学院, 2015.
[8]刘慧.国产大豆产量创十年来新高我国亟需打响“中国大豆”品牌[J].中国食品, 2018, 11:94-95
[9]Singh OV, Ghai S, Paul D, et al. Genetically modified crops:success, safety assessment, and public concern[J]. Appl Microbiol Biotechnol 2006, 71(5):598-607.
[10]崔宁波,张正岩.转基因大豆研究及应用进展[J].西北农业学报, 2016, 25(8):1111-1124.
[11]王宜林,李宏.抗除草剂转基因植物的研究现状[J].生物学通报, 2005, 40(4):15-17.
[12]郭兵福,等.不同大豆基因型对农杆菌敏感性研究及优异种质筛选[J].植物遗传资源学报, 2015, 16(3):569-575.
[13]王志刚,彭纯玉.中国转基因作物的发展现状与展望[J].农业展望, 2010, 6(11):51-55.
[14]沈泓,李超,李珏.分子生物学技术在转基因食品检测领域中的研究进展[J].中国农业信息, 2017, 15:57-59.
[15]Kamle M, et al. Current perspectives on genetically modified crops and detection methods[J]. Biotech, 2017, 7(3):219.
[16]Salisu IB, Shahid AA, Yaqoob A, et al. Molecular approaches for high throughput detection and quantification of genetically modified crops:A review[J]. Front Plant Sci 2017, 8:1670.
[17]Randhawa GJ, et al. Loop-mediated isothermal amplification:rapid visual and real-time methods for detection of genetically modified crops[J]. J Agric Food Chem 2013, 47:11338-11346.
[18]周杰,黄文胜,邓婷婷,等.环介导等温扩增法检测6种转基因大豆[J].农业生物技术学报, 2017, 25(2):335-344.
[19]吕蓓程,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学, 2010, 40(10):983-988.
[20]Piepenburg O, Williams CH, et al. DNA detection using recombination proteins[J]. PLoS Biology, 2006, 4(7):1115-1121.
[21]高威芳,朱鹏,黄海龙.重组酶聚合酶扩增技术:一种新的核酸扩增策略[J].中国生物化学与分子生物学报, 2016, 32(6):627-634.
[22]景志刚,董浩,狄栋栋,等.重组酶聚合酶扩增技术研究进展[J].生物技术通报, 2016, 32(6):47-53.
[23]周延培,孙海新,赵美丽,等.一种转基因马铃薯PCR鉴别方法的建立[J].粮油食品科技, 2018(3):67-70.
[24]Xu C, Li L, Jin W, Wan Y:Recombinase polymerase amplification(RPA)of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops[J]. Int J Mol Sci 2014,15(10):18197-18205.
[25]马建荣,余永红.市场销售大豆的转基因检测[J].广东化工,2018, 45(2):37-38.
[26]钱云开,高飞,王海洋,等. 16种转基因大豆品系的实时荧光PCR法筛查[J].中国油脂, 2017, 42(2):141-145.
[27]Liu M, Luo Y, Tao R, et al. Sensitive and Rapid Detection of Genetic Modified Soybean(Roundup Ready)by Loop-Mediated Isothermal Amplification[J]. Journal of the Agricultural Chemical Society of Japan 2009, 73(11):2365-2369.