多重PCR检测耐除草剂转基因作物
|
摘要
为建立耐除草剂转基因作物的高通量检测方法,本试验以目前生产上广泛应用的5种除草剂抗性基因dmo、pat、CP4EPSPS、bar和aad1为靶标进行多重PCR(MPCR)研究。通过引物适用性测试、反应体系中的不同引物浓度和反应程序中的退火温度测试、灵敏度和特异性验证等,建立了能同时检测5种除草剂抗性基因的MPCR检测方法。结果表明,当dmo、pat、CP4EPSPS、bar、aad1基因的检测引物终浓度分别为0.2、0.2、0.3、0.4、0.2μmol·L~(-1),退火温度为63℃时5种靶标扩增效果较好,且特异性条带清晰且均一。此外,MPCR检测方法具有较好的特异性,对每种靶标的检测灵敏度均可达到0.1%。适用性测试结果显示,MPCR检测方法可对含有5种除草剂抗性基因的多种转基因作物的单个品系或多个品系混合物进行筛选检测。无假阳性和假阴性结果表明,MPCR检测方法对实际样品具有很好的适用性。本试验结果为筛选高效耐除草剂转基因作物检测技术提供了一定的理论依据。
In order to establish a high-throughput detection method for herbicide-tolerant transgenic crops, five herbicide-tolerant genes, dmo, pat, CP4EPSPS, bar and aad1,which are widely used in production, were selected as targets in this study. By verifying the applicability of designed primers, determining the concentration of primers, annealing temperature, and the sensitivity and specificity of the detection system, the MPCR assay for detecting these five herbicide-tolerant genes simultaneously was developed. The results showed that when the final concentration of primers for dmo, pat, CP4EPSPS, bar and aad1 genes were 0.2, 0.2, 0.3, 0.4, 0.2 μmol·L~(-1), respectively, and the annealing temperature was 63℃, these five target genes could be well amplified and the specific bands were clear and uniform. In addition, the pentaplex PCR method had good specificity, and the detection sensitivity for each target gene could reach 0.1%. The applicability testing showed that this MPCR method could be used to screen the target genes from single event or mixture of several events, and there was no false positive and false negative amplification, which indicated that the system had good applicability in practical application. The study provided a high-efficient screening mean for the detection of herbicide tolerant genetically modified crops.
In order to establish a high-throughput detection method for herbicide-tolerant transgenic crops, five herbicide-tolerant genes, dmo, pat, CP4EPSPS, bar and aad1,which are widely used in production, were selected as targets in this study. By verifying the applicability of designed primers, determining the concentration of primers, annealing temperature, and the sensitivity and specificity of the detection system, the MPCR assay for detecting these five herbicide-tolerant genes simultaneously was developed. The results showed that when the final concentration of primers for dmo, pat, CP4EPSPS, bar and aad1 genes were 0.2, 0.2, 0.3, 0.4, 0.2 μmol·L~(-1), respectively, and the annealing temperature was 63℃, these five target genes could be well amplified and the specific bands were clear and uniform. In addition, the pentaplex PCR method had good specificity, and the detection sensitivity for each target gene could reach 0.1%. The applicability testing showed that this MPCR method could be used to screen the target genes from single event or mixture of several events, and there was no false positive and false negative amplification, which indicated that the system had good applicability in practical application. The study provided a high-efficient screening mean for the detection of herbicide tolerant genetically modified crops.
引文
[1] 国际农业生物技术应用服务组织. 2016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, 2017, 37(4): 1-8
[2] Clive J. Global Status of Commercialized Biotech/GM Crops[R]. Ithaca, NY: ISAAA, 2016 Brief No. 52
[3] 沈平, 武玉花, 梁晋刚, 卢新, 章秋艳, 王颢潜, 刘鹏程. 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] Qaim M, Kouser S. Genetically modified crops and food security[J]. PLoS One, 2013, 8(6): e64879
[5] Zhang D B, Guo J C. The development and standardization of testing methods for genetically modified organisms and their derived products[J]. Journal of Integrative Plant Biology, 2011, 53(7): 539-551
[6] Fraiture M A, Herman P, Taverniers Ⅰ, Loose M D, Deforce D, Roosens N H. Current and new approaches in GMO detection: challenges and solutions[J]. BioMed Research International, 2015, 4: 392872
[7] 张富丽, 雷绍荣, 刘勇. 转基因作物及加工品检测技术概述[J]. 生物技术通讯, 2009, 20(5): 733-737
[8] 谢家建,沈平,彭于发,李葱葱,宋贵文,孙爻. 转基因植物及其产品成分检测调控元件CaMV 35S 启动子、FMV 35S 启动子、NOS 启动子、NOS 终止子和CaMV 35S终止子定性PCR 方法[S]. 北京: 中国农业出版社,2012
[9] 瞿勇,武玉花,吴刚,曹应龙,卢长明. 转基因玉米MON88017 转化事件特异性定性PCR 检测方法及其标准化[J]. 农业生物技术学报, 2010,18(6): 1208-1214
[10] Germini A, Zanetti A, Salati C, Rossi S, Forré C, Schmid S, Marchelli R. Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods[J]. Journal of Agricultural and Food Chemistry, 2004, 52(11): 3275-3280
[11] 董立明, 邢珍娟, 李葱葱, 夏蔚, 闫伟, 李飞武. 应用多重PCR技术快速筛查食品中的转基因成分[J]. 食品与生物技术学报, 2017, 36(1): 56-61
[12] 黄银花, 胡晓湘, 徐慰倬, 高宇, 冯继东, 孙汉, 李宁. 影响多重PCR扩增效果的因素[J]. 遗传, 2003, 25(1): 65-68
[13] 张耀川, 许亮, 张洁, 田锦, 李玉舒, 石进朝. 利用多重PCR方法检测7种转基因卡诺拉油菜籽品种(系)研究[J]. 粮食与油脂, 2012(9): 30-32
[14] 董立明, 李葱葱, 邢珍娟, 邵改革, 夏蔚, 闫伟, 李飞武. 利用多重PCR技术快速检测五个转基因大豆品系[J]. 大豆科学, 2016, 35(6): 1002-1007
[15] 李飞武, 闫伟, 龙丽坤, 李葱葱, 张世宏, 张明. 应用多重PCR技术筛选检测转Bt基因作物[J]. 现代食品科技, 2014, 30(5): 262-266
[16] Xu W T, Zhai Z F, Huang K L, Zhang N, Yuan Y F, Shang Y, Luo Y B. A novel universal Primer-Multiplex-PCR method with sequencing gel electrophoresis analysis[J]. PLoS One, 2012, 7(1): e22900
[17] 傅凯, 黄文胜, 邓婷婷, 李富威, 刘昊, 陈颖. 多重PCR-液相芯片技术检测13个品系转基因玉米[J]. 中国食品学报, 2015, 15(1): 188-197
[18] Heide B R, Heir E, Holck A. Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis[J]. European Food Research and Technology, 2008, 227(2): 527-535
[19] Brinker R J, Burns W C, Feng P C, Gupta A, Hoi S W, Malven M, Wu K S. Soybean transgenic event MON87708 and methods of use thereof: US, US8501407[P]. 2013-08-06
[20] Cui Y X, Bryan J R, Maum D G, Wright T, Hamilton J L, Arnold N L, Vanopdorp N, Kaiser T M, Zhou N, Mcmaster S A, Peterson M A, Braxton L B, Gilles G J. AAD-1 event DAS-40278-9, related transgenic corn lines,and event-specific identification thereof, and methods of weed control involving AAD-1: US, US8598413[P]. 2013-12-03
[21] 农业部科技发展中心. 农业部1861号标准-5-2012: 转基因植物及其产品成分检测CP4-epsps基因定性PCR方法[S]. 北京: 中国农业出版社, 2012
[22] Datukishvili N, Kutateladze T, Gabriadze Ⅰ, Bitskinashvili K, Vishnepolsky B. New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 2015, 6: 757
[23] 尹全, 李忆, 宋君, 侯雪, 王东, 张富丽, 刘文娟, 常丽娟, 雷绍荣, 刘勇. 多重PCR筛查检测进口转基因玉米[J]. 核农学报, 2016, 30(6): 1045-1053
[24] 李会, 任志莹, 王颖, 王建忠. 多重PCR法快速检测转基因玉米多种转化体技术优势的比较分析[J]. 江苏农业科学, 2014, 42(5): 57-59
[25] 付伟, 任娇, 魏霜, 袁俊杰, 周广彪, 吴希阳, 朱水芳, 刘中勇. 转基因大豆DAS44406-6多重荧光定量PCR检测方法的建立[J]. 食品工业科技, 2017, 38(4): 63-66,72
[26] Kim J H, Jeong D, Kim Y R, Kwon Y K, Rhee G S, Zhang D B, Kim H Y. Development of a multiplex PCR method for testing six GM soybean events[J]. Food Control, 2013, 31(2): 366-371
[27] 向才玉, 凌杏园, 潘广, 康林, 陈冬美, 李秋枫, 刘新娇, 欧阳辉, 章桂明. 多重PCR-DHPLC法检测转基因棉花品系[J]. 植物检疫, 2015, 29(1): 37-41
[28] Li X, Wang X X, Yang J L, Liu Y M, He Y P, Pan L W. A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)[J]. BMC Biotechnology, 2014, 14: 43
[29] 白卫滨, 李宵, 朱翠娟, 文罗娜, 王丽芳, 卢军利, 欧仕益, 孙建霞. 通用引物多重PCR新技术对番木瓜转基因成分检测的研究[J]. 现代食品科技, 2016, 32(4): 229-234
[30] Guo J C, Chen L L, Liu X, Gao Y, Zhang D B. A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs[J]. Food Chemistry, 2012, 132: 1566-1573
[31] 柴帅杰, 武鹏程, 荣瑞娟, 郝育杰, 史佳楠, 郭亚璐, 丁国涛, 韩宇宁, 赵志静, 魏健, 尹长城, 刘国振. 转基因水稻中CP4-EPSPS蛋白质的检测及其表达特征研究[J]. 核农学报, 2017, 31(1): 44-50
[32] 农业部科技发展中心. 农业部1782号国家标准-6-2012: 转基因植物及其产品成分检测bar或pat基因定性PCR方法[S]. 北京: 中国农业出版社, 2012
[33] 龙丽坤, 韩月, 杨丹丹, 闫伟, 李飞武. 转基因作物中2种除草剂抗性基因aad1和dmo的PCR检测方法[J]. 生物安全学报, 2014, 23(4): 278-283
[2] Clive J. Global Status of Commercialized Biotech/GM Crops[R]. Ithaca, NY: ISAAA, 2016 Brief No. 52
[3] 沈平, 武玉花, 梁晋刚, 卢新, 章秋艳, 王颢潜, 刘鹏程. 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] Qaim M, Kouser S. Genetically modified crops and food security[J]. PLoS One, 2013, 8(6): e64879
[5] Zhang D B, Guo J C. The development and standardization of testing methods for genetically modified organisms and their derived products[J]. Journal of Integrative Plant Biology, 2011, 53(7): 539-551
[6] Fraiture M A, Herman P, Taverniers Ⅰ, Loose M D, Deforce D, Roosens N H. Current and new approaches in GMO detection: challenges and solutions[J]. BioMed Research International, 2015, 4: 392872
[7] 张富丽, 雷绍荣, 刘勇. 转基因作物及加工品检测技术概述[J]. 生物技术通讯, 2009, 20(5): 733-737
[8] 谢家建,沈平,彭于发,李葱葱,宋贵文,孙爻. 转基因植物及其产品成分检测调控元件CaMV 35S 启动子、FMV 35S 启动子、NOS 启动子、NOS 终止子和CaMV 35S终止子定性PCR 方法[S]. 北京: 中国农业出版社,2012
[9] 瞿勇,武玉花,吴刚,曹应龙,卢长明. 转基因玉米MON88017 转化事件特异性定性PCR 检测方法及其标准化[J]. 农业生物技术学报, 2010,18(6): 1208-1214
[10] Germini A, Zanetti A, Salati C, Rossi S, Forré C, Schmid S, Marchelli R. Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods[J]. Journal of Agricultural and Food Chemistry, 2004, 52(11): 3275-3280
[11] 董立明, 邢珍娟, 李葱葱, 夏蔚, 闫伟, 李飞武. 应用多重PCR技术快速筛查食品中的转基因成分[J]. 食品与生物技术学报, 2017, 36(1): 56-61
[12] 黄银花, 胡晓湘, 徐慰倬, 高宇, 冯继东, 孙汉, 李宁. 影响多重PCR扩增效果的因素[J]. 遗传, 2003, 25(1): 65-68
[13] 张耀川, 许亮, 张洁, 田锦, 李玉舒, 石进朝. 利用多重PCR方法检测7种转基因卡诺拉油菜籽品种(系)研究[J]. 粮食与油脂, 2012(9): 30-32
[14] 董立明, 李葱葱, 邢珍娟, 邵改革, 夏蔚, 闫伟, 李飞武. 利用多重PCR技术快速检测五个转基因大豆品系[J]. 大豆科学, 2016, 35(6): 1002-1007
[15] 李飞武, 闫伟, 龙丽坤, 李葱葱, 张世宏, 张明. 应用多重PCR技术筛选检测转Bt基因作物[J]. 现代食品科技, 2014, 30(5): 262-266
[16] Xu W T, Zhai Z F, Huang K L, Zhang N, Yuan Y F, Shang Y, Luo Y B. A novel universal Primer-Multiplex-PCR method with sequencing gel electrophoresis analysis[J]. PLoS One, 2012, 7(1): e22900
[17] 傅凯, 黄文胜, 邓婷婷, 李富威, 刘昊, 陈颖. 多重PCR-液相芯片技术检测13个品系转基因玉米[J]. 中国食品学报, 2015, 15(1): 188-197
[18] Heide B R, Heir E, Holck A. Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis[J]. European Food Research and Technology, 2008, 227(2): 527-535
[19] Brinker R J, Burns W C, Feng P C, Gupta A, Hoi S W, Malven M, Wu K S. Soybean transgenic event MON87708 and methods of use thereof: US, US8501407[P]. 2013-08-06
[20] Cui Y X, Bryan J R, Maum D G, Wright T, Hamilton J L, Arnold N L, Vanopdorp N, Kaiser T M, Zhou N, Mcmaster S A, Peterson M A, Braxton L B, Gilles G J. AAD-1 event DAS-40278-9, related transgenic corn lines,and event-specific identification thereof, and methods of weed control involving AAD-1: US, US8598413[P]. 2013-12-03
[21] 农业部科技发展中心. 农业部1861号标准-5-2012: 转基因植物及其产品成分检测CP4-epsps基因定性PCR方法[S]. 北京: 中国农业出版社, 2012
[22] Datukishvili N, Kutateladze T, Gabriadze Ⅰ, Bitskinashvili K, Vishnepolsky B. New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 2015, 6: 757
[23] 尹全, 李忆, 宋君, 侯雪, 王东, 张富丽, 刘文娟, 常丽娟, 雷绍荣, 刘勇. 多重PCR筛查检测进口转基因玉米[J]. 核农学报, 2016, 30(6): 1045-1053
[24] 李会, 任志莹, 王颖, 王建忠. 多重PCR法快速检测转基因玉米多种转化体技术优势的比较分析[J]. 江苏农业科学, 2014, 42(5): 57-59
[25] 付伟, 任娇, 魏霜, 袁俊杰, 周广彪, 吴希阳, 朱水芳, 刘中勇. 转基因大豆DAS44406-6多重荧光定量PCR检测方法的建立[J]. 食品工业科技, 2017, 38(4): 63-66,72
[26] Kim J H, Jeong D, Kim Y R, Kwon Y K, Rhee G S, Zhang D B, Kim H Y. Development of a multiplex PCR method for testing six GM soybean events[J]. Food Control, 2013, 31(2): 366-371
[27] 向才玉, 凌杏园, 潘广, 康林, 陈冬美, 李秋枫, 刘新娇, 欧阳辉, 章桂明. 多重PCR-DHPLC法检测转基因棉花品系[J]. 植物检疫, 2015, 29(1): 37-41
[28] Li X, Wang X X, Yang J L, Liu Y M, He Y P, Pan L W. A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)[J]. BMC Biotechnology, 2014, 14: 43
[29] 白卫滨, 李宵, 朱翠娟, 文罗娜, 王丽芳, 卢军利, 欧仕益, 孙建霞. 通用引物多重PCR新技术对番木瓜转基因成分检测的研究[J]. 现代食品科技, 2016, 32(4): 229-234
[30] Guo J C, Chen L L, Liu X, Gao Y, Zhang D B. A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs[J]. Food Chemistry, 2012, 132: 1566-1573
[31] 柴帅杰, 武鹏程, 荣瑞娟, 郝育杰, 史佳楠, 郭亚璐, 丁国涛, 韩宇宁, 赵志静, 魏健, 尹长城, 刘国振. 转基因水稻中CP4-EPSPS蛋白质的检测及其表达特征研究[J]. 核农学报, 2017, 31(1): 44-50
[32] 农业部科技发展中心. 农业部1782号国家标准-6-2012: 转基因植物及其产品成分检测bar或pat基因定性PCR方法[S]. 北京: 中国农业出版社, 2012
[33] 龙丽坤, 韩月, 杨丹丹, 闫伟, 李飞武. 转基因作物中2种除草剂抗性基因aad1和dmo的PCR检测方法[J]. 生物安全学报, 2014, 23(4): 278-283