环介导等温扩增技术在转基因杨树检测中的应用
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摘要
随着转基因技术的成熟,转基因生物越来越多,关于转基因生物的争议也越来越受到人们的关注。建立一套能够简便、快速、准确的检测转基因林木的外源基因技术,对转基因林木的研究有重要意义。采用灵敏、快速的环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,建立一套以Bt Cry1Ac或Npt II基因为靶标的转基因林木的检测方法。针对靶标基因的6个区域设计4种特异引物,优化LAMP-PCR反应体系中的各个成分,根据直接观察产物中焦磷酸镁的沉淀情况、Gold View核酸染色剂对其产物染色情况来判断反应结果并与将产物经过1%琼脂糖电泳的情况进行对比。比较LAMP-PCR和常规PCR两种不同检测方法的灵敏度。对转基因杨树不同无性系中多种外源基因进行检测。筛选出了最佳的引物,确定出了最优的反应体系:3 mmol/L Mg2+、1.4 mmol/L Bst、1.4 mol/L Betaine、7:1的10μmol/L内外引物比、2μL10 mmol/L d NTP、以及2μL浓度为200 ng/μL的DNA模板。在PCR仪65℃等温条件下保温90 min,可直接观察到焦磷酸镁的白色沉淀;经过Gold View核酸染色剂染色,可在紫外灯下观察到荧光绿色;经过1%琼脂糖电泳的检测,可观察到清晰的弥散状条带。通过对比试验得出LAMP-PCR的灵敏度比常规PCR要高。对实验室多个转基因杨树无性系目的基因的检测,证明了LAMP-PCR在转基因林木外源基因检测中的较高可靠性。本研究在国内外首次采用环介导等温扩增技术检测转基因林木,建立了一套更简便、灵敏、快速、可靠的转基因林木的LAMP-PCR检测方法。该技术在恒温65℃的条件下保温90 min即可完成转基因林木的检测,既节省了时间,又降低了成本,为转基因林木的检测研究提供了一种新的技术。
With the development of transgenic technology, the GM(genetically modified) organisms have been paid more and more debate and attention. It is important for the research of transgenic trees to develop a simple,rapid and accurate detection of transgenic trees of exogenous gene technology. A sensitive and rapid loop-mediated isothermal Amplification(LAMP) technique was used to establish the method to test transgenic forest trees with Bt Cry1Ac or Npt II gene as target. Four specific primers were designed in view of six regions of the target genes, to optimize the components of the LAMP-PCR reaction system. The reaction results are determined according to the results of direct observation of precipitation products in magnesium phosphate and staining of Gold View nucleic acid stain on the product. Then the products were compared through the 1% agarose electrophoresis. The sensitivity of two kinds of different detection methods of LAMP-PCR and common PCR were compared, and several exogenous genes were detected in different clones of transgenic poplar by the LAMP-PCR. The best primers were screened out, and the optimal reaction system was determined: 3 mmol/L Mg~(2+), 1.4 mmol/L Bst,1.4 mol/L Betaine, 7:1 10 μmol/L inner and outer primers, 2 μL 10 mmol/L d NTP and 2 μL 200 ng/μL DNA.The white precipitate of magnesium pyrophosphate can be directly observed under 65℃ condition for 90 min in the PCR apparatus; by Gold View nucleic acid stain, green fluorescence under can be observed under UV light;then detected by 1% agarose gel electrophoresis and clear diffuse bands can be observed. By contrast, the sensitivity of LAMP-PCR is higher than that of conventional PCR, and the subsequent verification test proves that LAMP-PCR has high reliability in the detection of a variety of target genes in the transgenic poplar clones. This research detected genetically modified trees with the LAMP technology at home and abroad for the first time, and established a simple, rapid, sensitive and reliable LAMP-PCR detection method of genetically modified trees. The technology completing the detection of genetically modified trees under the condition of constant temperature of 65℃ for 90 min that not only saved time but also reduced costs, provided a new technology for the detection of genetically modified trees research.
With the development of transgenic technology, the GM(genetically modified) organisms have been paid more and more debate and attention. It is important for the research of transgenic trees to develop a simple,rapid and accurate detection of transgenic trees of exogenous gene technology. A sensitive and rapid loop-mediated isothermal Amplification(LAMP) technique was used to establish the method to test transgenic forest trees with Bt Cry1Ac or Npt II gene as target. Four specific primers were designed in view of six regions of the target genes, to optimize the components of the LAMP-PCR reaction system. The reaction results are determined according to the results of direct observation of precipitation products in magnesium phosphate and staining of Gold View nucleic acid stain on the product. Then the products were compared through the 1% agarose electrophoresis. The sensitivity of two kinds of different detection methods of LAMP-PCR and common PCR were compared, and several exogenous genes were detected in different clones of transgenic poplar by the LAMP-PCR. The best primers were screened out, and the optimal reaction system was determined: 3 mmol/L Mg~(2+), 1.4 mmol/L Bst,1.4 mol/L Betaine, 7:1 10 μmol/L inner and outer primers, 2 μL 10 mmol/L d NTP and 2 μL 200 ng/μL DNA.The white precipitate of magnesium pyrophosphate can be directly observed under 65℃ condition for 90 min in the PCR apparatus; by Gold View nucleic acid stain, green fluorescence under can be observed under UV light;then detected by 1% agarose gel electrophoresis and clear diffuse bands can be observed. By contrast, the sensitivity of LAMP-PCR is higher than that of conventional PCR, and the subsequent verification test proves that LAMP-PCR has high reliability in the detection of a variety of target genes in the transgenic poplar clones. This research detected genetically modified trees with the LAMP technology at home and abroad for the first time, and established a simple, rapid, sensitive and reliable LAMP-PCR detection method of genetically modified trees. The technology completing the detection of genetically modified trees under the condition of constant temperature of 65℃ for 90 min that not only saved time but also reduced costs, provided a new technology for the detection of genetically modified trees research.
引文
Akihiro Y.,Shiori N.,Toshihiro A.,Masayo T.,Hiroaki K.,Hajime W.,Tsugunori N.,and Tadamichi T.,2005,Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis Tannerella forsythia,and Treponema denticola,Journal of Clinical Microbiology,43(5):2418-2424
Chen J.S.,Huang C.L.,Zhang X.H.,and Wu Z.Y.,2011Loop-mediated isothermal amplification for the detection of Ca MV35S promoter in genetically modified maize,Huabe Nongxuebao(Acta Agriculturae Boreali-Sinica),26(4):8-14(陈金松,黄丛林,张秀海,吴忠义,2011,环介导等温扩增技术检测含有Ca MV35S的转基因玉米,华北农学报,26(4):8-14)
Dai T.T.,and Wu X.Q.,2016,A method for rapidly identifying Phytophthora palmivora using the LAMP technique,Linye Kexue(Scientia Silvae Sinicae),52(10):161-166(戴婷婷吴小芹,2016,等温扩增技术快速检测棕榈疫,林业科学52(10):161-166)
Hiroki H.,Soichi K.,Satoru M.,Ken S.,Sadao O.,Yoshiyuki T.Seiji K.,Keiko T.,Keiko W.,Tsugunori N.,Hidenari Y.Sigenori M.,and Akira M.,2004,Rapid sexing of bovine preimplantation embryos using loop-mediated isotherma amplification,Theriogenology,62:887-896
Hiromi I.,Hiroko T.,Nona S.,Aya M.,Masami U.,Naoyuki I.Sugao O.,Noboru K.,Ikuo I.,and Takashi O.,2004,Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method,Journal of Clinica Microbiology,42(6):2465-2469
Liu J.J.,Wang G.Y.,Ren Y.C.,Dong Y.,Xu L.N.,and Liang HY.,2014,Genetic transformation and expression detection of tobacco transformed by Bt Cry1Ac and BADH bivalen gene,Hebei Linguo Yanjiu(Heibei Journal of Forestry and Orchard Research),29(2):169-174(刘娇娇,王桂英,任亚超,董研,徐丽娜,梁海永,2014,Bt Cry1Ac_BADH双价基因对烟草的遗传转化及表达检测,河北林果研究,29(2)169-174)
Manmohan P.,Kouhei H.,Hiroyuki I.,Paban K.D.,Parag S.Asha M.J.,Mohammed A.I.,Shingo I.,Norimitsu H.,and Kouichi M.,2005,Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay,Journa of Clinical Microbiology,43(6):2895-2903
Notomi T.,Okayama H.,Masubuchi H.,Yonekawa T.,Watanabe K.,Amino N.,and Hase T.,2000,Loop-mediated isotherma amplification of DNA,Nucleic Acids Res.,28(12):12
Pan H.,Duan Y.P.,Li M.L.,Chen F.M.,Fu Q.,and Zhang M.,2015,Sex identification of buffalo early embryo by LAMPmethod,Nanfang Nongye Xuebao(Journal of Southern Agriculture),46(6):1106-1110(潘宏,段亚萍,李敏玲,陈富美,富强,张明,2015,LAMP法在水牛早期胚胎性别鉴定中的应用,南方农业学报,46(6):1106-1110)
Shen H.P.,2012,The study on the detection of genetically modified soybean and soybean products using loop-mediated isothermal amplification,Thesis for M.S.,South China University of Technology,Supervisor:Shi L.,pp.25-26(沈会平,2012,环介导等温扩增法检测转基因大豆及其制品的研究,硕士学位论文,华南理工大学,导师:石磊,pp.25-26)
Tomotada I.,Toshiaki S.,and Kozaburo H.,2003,Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex,M.avium,and M.intracellulare in sputum samples,Journal of Clinical Microbiology,41(6):2616-2622
Wang G.L.,Qiu Y.X.,and Fan H.Y.,2015,Establishment and application of PCR detection system of genetically modified maize in laboratory,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1778-1783(王国莉,丘裕雄,范红英,2015,转基因玉米实验室PCR检测体系的优化及应用,基因组学与应用生物学,34(8):1778-1783)
Wang J.,and He P.C.,2000,Extraction of genomic DNA from vitis amurensis and its RAPD analysis,Guoshu Kexue(Journal of Fruit Science),17(2):79-82(王军,贺普超,2000,山葡萄基因组DNA提取及RAPD鉴定,果树科学,17(2):79-82)
Wang Y.,2009,Development and application of loop-mediated isothermal amplification for detection of genetically modified crops,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(4):1473-1477(王永,2009,转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用,中国农业科学,42(4):1473-1477)
Wu Y.X.,Li P.J.,Wang J.,and Yang F.L.,2016,Loop-mediated isothermal amplification method and its application in pathogen detection,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),43(2):389-393(吴禹熹,李璞君,王娟,杨发龙,2016,LAMP方法及其在病原微生物检测中的应用,中国畜牧兽医,43(2):389-393)
Yasuyoshi M.,Kentaro N.,Norihiro T.,and Tsugunori N.,2001,Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation,Biochemical and Biophysical Research Communications,289(1):150-154
Zhang F.,2016,Rapid detection of tra nsgenic crops based on DNA isothermal amplification,Dissertation for Ph.D.,Zhejiang University,Supervisor:Wu J.,and Ying Y.B.,pp.1-8(张芳,2016,基于DNA恒温扩增技术的转基因作物快速检测方法研究,博士学位论文,浙江大学,导师:吴坚,应义斌,pp.1-8)
Zhou L.H.,2012,The study on the detection of genetically modi-fied in foods using loop-mediated isothermal amplification,Thesis for M.S.,Southern Medical University,Supervisor:Ma W.L.,pp.61-63(周琳华,2012,LAMP法在转基因食品检测中的应用研究,硕士学位论文,南方医科大学,导师:马文丽,pp.61-63)
Chen J.S.,Huang C.L.,Zhang X.H.,and Wu Z.Y.,2011Loop-mediated isothermal amplification for the detection of Ca MV35S promoter in genetically modified maize,Huabe Nongxuebao(Acta Agriculturae Boreali-Sinica),26(4):8-14(陈金松,黄丛林,张秀海,吴忠义,2011,环介导等温扩增技术检测含有Ca MV35S的转基因玉米,华北农学报,26(4):8-14)
Dai T.T.,and Wu X.Q.,2016,A method for rapidly identifying Phytophthora palmivora using the LAMP technique,Linye Kexue(Scientia Silvae Sinicae),52(10):161-166(戴婷婷吴小芹,2016,等温扩增技术快速检测棕榈疫,林业科学52(10):161-166)
Hiroki H.,Soichi K.,Satoru M.,Ken S.,Sadao O.,Yoshiyuki T.Seiji K.,Keiko T.,Keiko W.,Tsugunori N.,Hidenari Y.Sigenori M.,and Akira M.,2004,Rapid sexing of bovine preimplantation embryos using loop-mediated isotherma amplification,Theriogenology,62:887-896
Hiromi I.,Hiroko T.,Nona S.,Aya M.,Masami U.,Naoyuki I.Sugao O.,Noboru K.,Ikuo I.,and Takashi O.,2004,Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method,Journal of Clinica Microbiology,42(6):2465-2469
Liu J.J.,Wang G.Y.,Ren Y.C.,Dong Y.,Xu L.N.,and Liang HY.,2014,Genetic transformation and expression detection of tobacco transformed by Bt Cry1Ac and BADH bivalen gene,Hebei Linguo Yanjiu(Heibei Journal of Forestry and Orchard Research),29(2):169-174(刘娇娇,王桂英,任亚超,董研,徐丽娜,梁海永,2014,Bt Cry1Ac_BADH双价基因对烟草的遗传转化及表达检测,河北林果研究,29(2)169-174)
Manmohan P.,Kouhei H.,Hiroyuki I.,Paban K.D.,Parag S.Asha M.J.,Mohammed A.I.,Shingo I.,Norimitsu H.,and Kouichi M.,2005,Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay,Journa of Clinical Microbiology,43(6):2895-2903
Notomi T.,Okayama H.,Masubuchi H.,Yonekawa T.,Watanabe K.,Amino N.,and Hase T.,2000,Loop-mediated isotherma amplification of DNA,Nucleic Acids Res.,28(12):12
Pan H.,Duan Y.P.,Li M.L.,Chen F.M.,Fu Q.,and Zhang M.,2015,Sex identification of buffalo early embryo by LAMPmethod,Nanfang Nongye Xuebao(Journal of Southern Agriculture),46(6):1106-1110(潘宏,段亚萍,李敏玲,陈富美,富强,张明,2015,LAMP法在水牛早期胚胎性别鉴定中的应用,南方农业学报,46(6):1106-1110)
Shen H.P.,2012,The study on the detection of genetically modified soybean and soybean products using loop-mediated isothermal amplification,Thesis for M.S.,South China University of Technology,Supervisor:Shi L.,pp.25-26(沈会平,2012,环介导等温扩增法检测转基因大豆及其制品的研究,硕士学位论文,华南理工大学,导师:石磊,pp.25-26)
Tomotada I.,Toshiaki S.,and Kozaburo H.,2003,Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex,M.avium,and M.intracellulare in sputum samples,Journal of Clinical Microbiology,41(6):2616-2622
Wang G.L.,Qiu Y.X.,and Fan H.Y.,2015,Establishment and application of PCR detection system of genetically modified maize in laboratory,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1778-1783(王国莉,丘裕雄,范红英,2015,转基因玉米实验室PCR检测体系的优化及应用,基因组学与应用生物学,34(8):1778-1783)
Wang J.,and He P.C.,2000,Extraction of genomic DNA from vitis amurensis and its RAPD analysis,Guoshu Kexue(Journal of Fruit Science),17(2):79-82(王军,贺普超,2000,山葡萄基因组DNA提取及RAPD鉴定,果树科学,17(2):79-82)
Wang Y.,2009,Development and application of loop-mediated isothermal amplification for detection of genetically modified crops,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),42(4):1473-1477(王永,2009,转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用,中国农业科学,42(4):1473-1477)
Wu Y.X.,Li P.J.,Wang J.,and Yang F.L.,2016,Loop-mediated isothermal amplification method and its application in pathogen detection,Zhongguo Xumu Shouyi(China Animal Husbandry&Veterinary Medicine),43(2):389-393(吴禹熹,李璞君,王娟,杨发龙,2016,LAMP方法及其在病原微生物检测中的应用,中国畜牧兽医,43(2):389-393)
Yasuyoshi M.,Kentaro N.,Norihiro T.,and Tsugunori N.,2001,Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation,Biochemical and Biophysical Research Communications,289(1):150-154
Zhang F.,2016,Rapid detection of tra nsgenic crops based on DNA isothermal amplification,Dissertation for Ph.D.,Zhejiang University,Supervisor:Wu J.,and Ying Y.B.,pp.1-8(张芳,2016,基于DNA恒温扩增技术的转基因作物快速检测方法研究,博士学位论文,浙江大学,导师:吴坚,应义斌,pp.1-8)
Zhou L.H.,2012,The study on the detection of genetically modi-fied in foods using loop-mediated isothermal amplification,Thesis for M.S.,Southern Medical University,Supervisor:Ma W.L.,pp.61-63(周琳华,2012,LAMP法在转基因食品检测中的应用研究,硕士学位论文,南方医科大学,导师:马文丽,pp.61-63)