转基因大豆MON89788实时荧光重组酶聚合酶扩增检测方法的建立
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摘要
MON89788是较早被批准商业化种植的转基因大豆(Glycine max)品种,种植范围广,产品流通量大。本研究利用重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA),针对MON89788的转化体特异性序列设计了引物和探针,利用正反向引物筛选的方法获得了最佳引物组合,并对反应的温度、引物和探针的浓度进行了优化选择。结果表明,RPA在29.5～43.1℃范围内都能扩增,对温度的容忍度较大,高浓度的探针会影响实时荧光RPA的扩增效率。同时对实时荧光RPA检测体系的特异性、灵敏度和适用性等进行测试,最终建立了MON89788实时荧光RPA检测方法。该检测方法特异性强,对MON89788的绝对检测限(absolute limit of detection, aLOD)可达到40拷贝,相对检测限(relative limit of detection, rLOD)为0.05%,对实际样品的检测在39℃,10 min内完成,是实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测时间的0.07～0.13倍。该恒温、快速的检测方法为转基因成分的快速检测提供了新的技术支持,有望用于转基因成分的现场快速检测。
Transgenic soybean(Glycine max) MON89788 is approved for commercial planting earlier. It has a wide range of cultivation with a large circulation of products. In this study, a series of primers combining with a special probe were designed for recombinase polymerase amplification(RPA) based on the event-specific sequence of MON89788. Then a strategy that forward primers and reverse primers mutually amplifying was employed to obtain the optimal primers combination with the highest amplification efficiency. In addition, the RPA reaction conditions, including reaction temperature, the concentration of primers and probe, were selected and optimized. The results indicated that RPA had a wide range of amplification temperature: Among 29.5～43.1 ℃, and the high concentration of probe in the reaction system would affect the amplification efficiency of RPA. Further, the specificity, sensitivity and applicability of the RPA detection system were tested. Then the MON89788 real-time fluorescent RPA(RT-RPA) detection method was established. The detection method was specific, and the absolute limit of detection(aLOD) of MON89788 could reach 40 copies, the relative limit of detection(rLOD) was 0.05%. Furthermore, for real samples detection, the RT-RPA detection was completed within 10 min at 39 ℃, which was 0.07～0.13 times of quantitative real-time PCR(qRT-PCR) detection time.This isothermal and rapid detection method provides new technical support for the rapid detection of genetically modified components, and is expected to be used for rapid on-site detection of genetically modified components.
Transgenic soybean(Glycine max) MON89788 is approved for commercial planting earlier. It has a wide range of cultivation with a large circulation of products. In this study, a series of primers combining with a special probe were designed for recombinase polymerase amplification(RPA) based on the event-specific sequence of MON89788. Then a strategy that forward primers and reverse primers mutually amplifying was employed to obtain the optimal primers combination with the highest amplification efficiency. In addition, the RPA reaction conditions, including reaction temperature, the concentration of primers and probe, were selected and optimized. The results indicated that RPA had a wide range of amplification temperature: Among 29.5～43.1 ℃, and the high concentration of probe in the reaction system would affect the amplification efficiency of RPA. Further, the specificity, sensitivity and applicability of the RPA detection system were tested. Then the MON89788 real-time fluorescent RPA(RT-RPA) detection method was established. The detection method was specific, and the absolute limit of detection(aLOD) of MON89788 could reach 40 copies, the relative limit of detection(rLOD) was 0.05%. Furthermore, for real samples detection, the RT-RPA detection was completed within 10 min at 39 ℃, which was 0.07～0.13 times of quantitative real-time PCR(qRT-PCR) detection time.This isothermal and rapid detection method provides new technical support for the rapid detection of genetically modified components, and is expected to be used for rapid on-site detection of genetically modified components.
引文
黄新,朱水芳,高宏伟,等.2016.植物及其加工产品中转基因成分实时荧光PCR定性检验方法[S].SN/T 1204-2016.(Huang X,Zhu S F,Gao H W,et al.2016.Protocol of the real-time PCR method for detecting genetically modified plants and their derived products[S].SN/T1204-2016.)
杨华,彭城,肖英平,等.2018.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,26(3):492-501.(Yang H,Peng C,Xiao Y P,et al.2018.Study of conventional PCR and qRT-PCRdetection methods for genetically modified soybean(Glycine max)SHZD32-1[J].Journal of Agricultural Biotechnology,26(3):492-501.)
杨阳,王叶,范金杰,等.2016.转基因棉花MON757转化体特异性PCR检测方法及应用[J].农业生物技术学报,24(6):908-918.(Yang Y,Wang Y,Fan J J,et al.2016.Event-specific PCR detection methods of genetically modified cotton(Gossypium hirsutum)MON757 and their application[J].Journal of Agricultural Biotechnology,24(6):908-918.)
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European network of GMO laboratories(ENGL).2008.Definition of minimum performance requirements for analytical methods of GMO testing.GM Food and Feed[Z],1-8.
Gressel J.2010.Needs for and environmental risks from transgenic crops in the developing world[J].New Biotechnology,27(5):522-527.
Holst-Jensen A.2009.Testing for genetically modified organisms(GMOs):Past,present and future perspectives[J].Biotechnology Advances,27(6):1071-1082.
ISAAA.2017.Global Status of Commercialized Biotech/GMCrops in 2017:Biotech Crop Adoption Surges as Economic Benefits Accumulate in 22 Years.ISAAA Brief No.53.ISAAA:Ithaca,NY.(http://www.isaaa.org/resources/publications/briefs/53/download/isaaa-brief-53-2017.pdf)
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Mayboroda O,Benito A G,del Rio,Jonathan Sabaté,et al.2016.Isothermal solid-phase amplification system for detection of Yersinia pestis[J].Analytical and Bioanalytical Chemistry,408(3):671-676.
Panda R,Ariyarathna H,Amnuaycheewa P,et al.2013.Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety[J].Allergy,68(2):142-151.
Piepenburg O,Williams C,Stemple D N.2006.DNA detection using recombination proteins[J].PLoS Biology,4(7):1115-1121.
Rijssen W J V,Morris E J.2018.Chapter 13-safety and risk assessment of food from genetically engineered crops and animals:The challenges[J].Genetically Engineered Foods,335-368.
Rui W,Fang Z,Liu W,et al.2017.Instant,visual,and instrument-free method for on-site screening of GTS 40-3-2soybean based on body-heat triggered recombinase polymerase amplification[J].Analytical Chemistry,89(8):4413-4418.
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.
Yamanaka E S,Tortajadagenaro L A,ángel Maquieira.2017.Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection[J].Microchimica Acta,184(5):1453-1462.
Zipper H,Brunner H,Bernhagen J,et al.2014.Investigations on DNA intercalation and surface binding by SYBR Green I,its structure determination and methodological implications[J].Nucleic Acids Research,32(12):e103.
杨华,彭城,肖英平,等.2018.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,26(3):492-501.(Yang H,Peng C,Xiao Y P,et al.2018.Study of conventional PCR and qRT-PCRdetection methods for genetically modified soybean(Glycine max)SHZD32-1[J].Journal of Agricultural Biotechnology,26(3):492-501.)
杨阳,王叶,范金杰,等.2016.转基因棉花MON757转化体特异性PCR检测方法及应用[J].农业生物技术学报,24(6):908-918.(Yang Y,Wang Y,Fan J J,et al.2016.Event-specific PCR detection methods of genetically modified cotton(Gossypium hirsutum)MON757 and their application[J].Journal of Agricultural Biotechnology,24(6):908-918.)
中华人民共和国农业部.2002.农业转基因生物标识管理办法[Z].北京:中华人民共和国农业部.Ministry of Agriculture of the People's Republic of China.2002.Measures for the administration of GMO labeling in agricultur[Z].Beijing:Ministry of Agriculture of the People's Republic of China
European network of GMO laboratories(ENGL).2008.Definition of minimum performance requirements for analytical methods of GMO testing.GM Food and Feed[Z],1-8.
Gressel J.2010.Needs for and environmental risks from transgenic crops in the developing world[J].New Biotechnology,27(5):522-527.
Holst-Jensen A.2009.Testing for genetically modified organisms(GMOs):Past,present and future perspectives[J].Biotechnology Advances,27(6):1071-1082.
ISAAA.2017.Global Status of Commercialized Biotech/GMCrops in 2017:Biotech Crop Adoption Surges as Economic Benefits Accumulate in 22 Years.ISAAA Brief No.53.ISAAA:Ithaca,NY.(http://www.isaaa.org/resources/publications/briefs/53/download/isaaa-brief-53-2017.pdf)
Ivan M L,Ciara K O.2018.Recombinase polymerase amplification:Basics,applications and recent advances[J].Trends in Analytical Chemistry,98:19-35
Mayboroda O,Benito A G,del Rio,Jonathan Sabaté,et al.2016.Isothermal solid-phase amplification system for detection of Yersinia pestis[J].Analytical and Bioanalytical Chemistry,408(3):671-676.
Panda R,Ariyarathna H,Amnuaycheewa P,et al.2013.Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety[J].Allergy,68(2):142-151.
Piepenburg O,Williams C,Stemple D N.2006.DNA detection using recombination proteins[J].PLoS Biology,4(7):1115-1121.
Rijssen W J V,Morris E J.2018.Chapter 13-safety and risk assessment of food from genetically engineered crops and animals:The challenges[J].Genetically Engineered Foods,335-368.
Rui W,Fang Z,Liu W,et al.2017.Instant,visual,and instrument-free method for on-site screening of GTS 40-3-2soybean based on body-heat triggered recombinase polymerase amplification[J].Analytical Chemistry,89(8):4413-4418.
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.
Yamanaka E S,Tortajadagenaro L A,ángel Maquieira.2017.Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection[J].Microchimica Acta,184(5):1453-1462.
Zipper H,Brunner H,Bernhagen J,et al.2014.Investigations on DNA intercalation and surface binding by SYBR Green I,its structure determination and methodological implications[J].Nucleic Acids Research,32(12):e103.
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