转基因作物核酸快速提取和检测方法研究
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摘要
随着越来越多的转基因作物的发展和商业化种植,转基因作物核酸快速提取及其检测技术的研究已经变得越来越必要。LAMP(Loop-mediated isothermal amplification)是一种环介导的等温扩增方法,比较传统的聚合酶链式反应,它具有特异性高、简单、快速等特点,不需要精密昂贵的仪器(如:PCR仪)和繁琐凝胶电泳检测。由于实现了可视化的检测,在60分钟内就能完成检测,大大缩短了检测时间,适合转基因植物的现场快速检测。另外,现有的提取转基因作物的DNA方法所耗用的时间在90分钟左右,同时需要笨重的离心机作为提取时必需的仪器,我们研究了一种快速提取DNA的技术,可以将抽提DNA的时间由现有的90分钟左右缩短到10-15分钟左右。将快速提取DNA技术与快速检测LAMP方法相结合,就能在90分钟完成提取和检测转基因作物的整个过程,实现快速现场检测
1、根据七种转基因玉米品系(DAS-59122-7, T25, BT176, TC1507, MON810, BT11 and MON863)的旁邻序列,设计品系特异性引物,优化LAMP反应体系,代替凝胶电泳方法,通过加入高浓度的SYBR Green I荧光染料的可视化方法来检测LAMP的产物。建立七种转基因玉米品系特异性检测的LAMP方法,同时将这种方法应用于实际样品的检测。实验结果证明建立的七种转基因玉米LAMP检测方法具有高特异性,除了MON810品系以外灵敏度都可以达到4个拷贝数的基因组DNA。并且将已经建立的检测方法运用于GIPSA(美国谷物检验、批发及畜牧场管理局)样品的检测,检测结果与最后的GIPSA的最终报告中结果相一致;
2、本文研究转基因生物快速现场提取DNA技术,设计一种简单快速提取DNA的装置。这种技术采用气压法代替离心的方法,用简单的注射器的气压提取DNA的方法,代替笨重的离心机,将抽提DNA的时间由现有的90分钟左右缩短到10-15分钟左右,应用这种方法提取水稻,玉米,棉花,大豆的DNA,实现野外快速现场提取转基因作物的DNA。同时结合LAMP方法实现快速现场检测的目的。
As more and more GM crops were approved for commercialization and planting, the development of quick and on-spot analysis methods for GM crops and their derivates are very important and useful. Loop mediated isothermal amplification (LAMP) is one isothermal nucleic acids amplification technique. The LAMP reaction shows the higher specificity, simplicity, rapidness, compared with typical PCR. It is no need of the expensive PCR equipments and argarose gel electrophoresis (AGE) assays. The technique is less-consuming time for DNA amplification (within 60 min) and high efficiency by direct visual inspection, which can be used for large numbers of GMOs detection on spot. Furthermore, the existing extraction DNA methods for transgenic crops are consuming time(about 90 min). Simultaneously, it is required for the unwieldy centrifuge instrument taking the extraction. We studied a novel technology for extracting of DNA to reduce the time from the existing about 90 minutes to about 10-15 minutes. Combined the fast extracting DNA technology with the LAMP method, the extraction and detection can be completed within 90 minutes for on spot detection of GMOs.
1、Based on the event-specific sequences of seven GM maizes (DAS-59122-7, T25, BT176, TC1507, MON810, BT11 and MON863), LAMP event-specific primers were designed using the specific software of Primer Explorer V4. In this study, the optimized visual LAMP methods for the detection of GM maize events have been established. In combination with SYBR Green I dye, the amplified products can be directly detected by naked eye instead of the conventional gel electropherisis anlaysis. The limits of detection (LODs) were as low as four copies of maize haploid genomic DNA except for the forty copies of MON810 assay. Furthermore, these developed assays were successfully used in the USDA/GIPSA Proficiency Program, all the six mixed maize samples with different contents (ranged from 0.0% to 2.0%) of GM maize events were identified correctly. The LAMP assay has the advantages of time-saving and low cost or simple procedures for result analysis, and can be used for GMOs detection on spot.
2、Research on the rapid extraction of DNA for GM organism. We designed simple equipment for extracting DNA, which contains a simple injector instead of the unwieldy centrifuge instrument. The technology used principle of atmospheric pressure, instead of weightlessness. We studied the novel technology for extracting DNA to reduce the time from the existing about 90 minutes to about 10-15 minutes. The technology was employed to extract the rice, the maize, the cotton and the soybean, and was used to fast extraction on spot. Combined the fast extracting DNA technology with the LAMP method, the method can be used for GMOs detection on spot.
1、根据七种转基因玉米品系(DAS-59122-7, T25, BT176, TC1507, MON810, BT11 and MON863)的旁邻序列,设计品系特异性引物,优化LAMP反应体系,代替凝胶电泳方法,通过加入高浓度的SYBR Green I荧光染料的可视化方法来检测LAMP的产物。建立七种转基因玉米品系特异性检测的LAMP方法,同时将这种方法应用于实际样品的检测。实验结果证明建立的七种转基因玉米LAMP检测方法具有高特异性,除了MON810品系以外灵敏度都可以达到4个拷贝数的基因组DNA。并且将已经建立的检测方法运用于GIPSA(美国谷物检验、批发及畜牧场管理局)样品的检测,检测结果与最后的GIPSA的最终报告中结果相一致;
2、本文研究转基因生物快速现场提取DNA技术,设计一种简单快速提取DNA的装置。这种技术采用气压法代替离心的方法,用简单的注射器的气压提取DNA的方法,代替笨重的离心机,将抽提DNA的时间由现有的90分钟左右缩短到10-15分钟左右,应用这种方法提取水稻,玉米,棉花,大豆的DNA,实现野外快速现场提取转基因作物的DNA。同时结合LAMP方法实现快速现场检测的目的。
As more and more GM crops were approved for commercialization and planting, the development of quick and on-spot analysis methods for GM crops and their derivates are very important and useful. Loop mediated isothermal amplification (LAMP) is one isothermal nucleic acids amplification technique. The LAMP reaction shows the higher specificity, simplicity, rapidness, compared with typical PCR. It is no need of the expensive PCR equipments and argarose gel electrophoresis (AGE) assays. The technique is less-consuming time for DNA amplification (within 60 min) and high efficiency by direct visual inspection, which can be used for large numbers of GMOs detection on spot. Furthermore, the existing extraction DNA methods for transgenic crops are consuming time(about 90 min). Simultaneously, it is required for the unwieldy centrifuge instrument taking the extraction. We studied a novel technology for extracting of DNA to reduce the time from the existing about 90 minutes to about 10-15 minutes. Combined the fast extracting DNA technology with the LAMP method, the extraction and detection can be completed within 90 minutes for on spot detection of GMOs.
1、Based on the event-specific sequences of seven GM maizes (DAS-59122-7, T25, BT176, TC1507, MON810, BT11 and MON863), LAMP event-specific primers were designed using the specific software of Primer Explorer V4. In this study, the optimized visual LAMP methods for the detection of GM maize events have been established. In combination with SYBR Green I dye, the amplified products can be directly detected by naked eye instead of the conventional gel electropherisis anlaysis. The limits of detection (LODs) were as low as four copies of maize haploid genomic DNA except for the forty copies of MON810 assay. Furthermore, these developed assays were successfully used in the USDA/GIPSA Proficiency Program, all the six mixed maize samples with different contents (ranged from 0.0% to 2.0%) of GM maize events were identified correctly. The LAMP assay has the advantages of time-saving and low cost or simple procedures for result analysis, and can be used for GMOs detection on spot.
2、Research on the rapid extraction of DNA for GM organism. We designed simple equipment for extracting DNA, which contains a simple injector instead of the unwieldy centrifuge instrument. The technology used principle of atmospheric pressure, instead of weightlessness. We studied the novel technology for extracting DNA to reduce the time from the existing about 90 minutes to about 10-15 minutes. The technology was employed to extract the rice, the maize, the cotton and the soybean, and was used to fast extraction on spot. Combined the fast extracting DNA technology with the LAMP method, the method can be used for GMOs detection on spot.
引文
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[3] Jonas, DA. Antignac, E. Antoine, JM. The safety assessment of novel foods. Guidelines prepared by ILSI Europe Novel Food Task Force. Food Chem Toxicol, 1996, 34, 931-940.
[4] IFBC. Biotechnologies and food: assuring the safety of foods produced by genetic Modification. Regul Toxicol Pharmacol, 1990,12, 1-196.
[5] Commission Decision 97/98/EC of 23 January 1997 concerning the placing on the market of genetically modified maize (Zea mays L.) with the combined modification for insecticidal properties conferred by the Bt-endotoxin gene and increased tolerance to the herbicide glufosinate ammonium pursuant to Council Directive 90/220/EEC. Official Journal. 1997, L031, 0069-0070.
[6] Council Regulation (EC) No 1139/98,concerning the compulsory indication of the labelling of certain foodstuffs produced from genetically modified organisms of particulars other than those provided for in Directive 79/112/EEC. Official Journal.1998, L159, 0004-0007.
[7] Commission Regulation (EC) No 49/2000 of 10 January 2000 amending Council regulation (EC) No 1139/98 concerning the compulsory indication on the labelling of certain foodstuffs produced from genetically modified organisms of particulars other than those provided for in Directive 79/112/EEC. Official Journal. 2000, L006, 0013-0014.
[8] European Commission, Question and Answers on the regulation of GMOs in the EU, MEMO/02/160, Brussels, 15 October 2002.
[9] European Parliament and Council, Regulation (EC) N. 178/2002 of 28 January 2002, laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Official Journal. 2002, L31.
[10] European Parliament and Council, Regulation (EC) N. 1829/2003, genetically modified food and feed; Official Journal. 2003, L268, 1.
[11] European Parliament and Council, Regulation (EC) N. 1830/2003, 2003, concerning the traceability and labelling of genetically modified organisms and the traceability of food and feedproducts from genetically modified organisms and amending Directive 2001/18/EC. Official Journal. 2003. L268, 24.
[12] Commission Decision 96/281/EC of 3 April 1996 concerning the placing on the market of genetically modified soya beans (Glycine max L.) with increased tolerance to the herbicide glyphosate, pursuant to Council Directive 90/220/EEC. Official Journal. 1996, L107, 0010-0011.
[13] Ministry of Agriculture Decree No. 10 of China (2001). Ministry of Agriculture, People's Republic of China: Labeling management on agricultural genetically modified organisms (中华人民共和国农业部10号令《农业转基因生物标识管理办法》).
[14] General Guidelines on Sampling, CAC/GL 50, 2004.
[15] Guide on Sampling for Analysis of Foods, NMKL Procedure N12, 2002
[16] http://www.shgmo.org/images/UserFunction/process.jpg
[17] Anklam E, Gadani F, Heinze P, Pijnenburg H, Van den Eede G (2002) Analytical methods for Detection and determination of genetically modified organisms (GMOs) in agricultural crops and plant-derived food products. Eur Food Res Technol. 214, 3-26.
[18] Lipp M, Shillito R, Giroux R, Spiegelhalter F, Charlton S, Pinero D, Song P (2005) Polymerase chain reaction technology as an analytical tool in agricultural biotechnology. J AOAC Int. 88, 136-155.
[19] Kleppe K, Ohtsuka E, Kleppe R. et al. Studies on polynucleotides: Repair replication of short synthetic DNAs as catalysed by DNA polymerase. J Mol Biol. 1971, 56, 341-361.
[20] Ahmed, FE. Detection of genetically modified organisms in foods. Trends in Biotechnology. 2002, 20, 215-223.
[21] WELSH J,MMCLELLAN M. Fingerprinting Genomes Using PCR with Arbitary Primers[J]. Nucleic Acids Research. 1990, 18, 72l3-72l8.
[22] http://www.shgmo.org/pages/protect/user/detect_service_method
[23] LT Yang, SC Xu, AH Pai, CS Yin, KW Zhang, ZY Wang, ZG Zhou, and DB Zhang. Event Specific Qualitative and Quantitative Polymerase Chain Reaction Detection of Genetically Modified MON863 Maize Based on the 5￠-Transgene Integration Sequence. J. Agric. Food Chem. 2005, 53, 9312-9318.
[24] Holck A, Va?¨tilingom M, Didierjean L, Rudi K. 5￠Nuclease PCR for quantitative event specific detection of the genetically modified MON810 MaisGard maize. Eur. Food Res. Technol. 2002,214, 449-454.
[25] R?nning SB, Va?¨tilingom M, Berdal KG, Holst-Jensen, A. Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays). Eur. Food Res. Technol. 2003, 216, 347-354.
[26] Hernandez M, Esteve T, Prat S, Pla M. Development of real-time PCR systems based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J. Cereal Sci. 2004, 39, 99-107.
[27] Huang HY, Pan TM. Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods. J Agric Food Chem. 2004, 52, 3264-3268.
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