摘要
快速准确的转基因检测技术是严格监管转基因作物及其产品的重要支撑;不同转基因作物由于导入不同外源目的基因从而表现出不同性状,因此外源目的基因序列可作为基于核酸检测技术进行转基因筛选的标志物.基于环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,以中性红染料作为比色试剂,建立玉米内源基因和玉米转基因成分的基因特异性的可视化快速检测方法.针对玉米内源基因IVR基因,抗虫外源bar、pat基因,和Bt176中的抗除草剂外源cry1Ab基因分别设计了LAMP引物.结果表明,在Real-time LAMP体系下,能够实现对玉米内源IVR基因、转基因玉米品系Bt176中的bar基因和转基因玉米品系TC1507和59122中的pat基因的检测.对转基因玉米品系Bt176中的cry1Ab基因设计的引物能特异性扩增Bt176,而对Bt11和MON810两种转基因玉米品系无效.通过优化Tris-HCl和中性红浓度,在5 mmol/L Tris-HCl和100μmol/L中性红浓度条件下建立了可视化检测体系,反应1 h即可分别实现对玉米内源基因IVR基因,抗虫外源bar、pat基因,和抗除草剂外源cry1Ab基因的检测.中性红可视化检测结果与Real-time LAMP荧光扩增曲线结果一致,体现了该可视化检测方法具有较高的准确性.本研究建立了一种对玉米转基因相关成分的可视化检测体系,该方法既快速又简便且尤其适用于现场检测.(图6表1参17).
Owing to the rapid development and application of the genetic modification technology,strict supervision of genetically modified(GM)crops and their products is imperative.To achieve this goal,it is important to develop a rapid and accurate GMO testing technology to serve as a supporting requirement.GM crops often contain different exogenous genes for various transgenic resistances and they can be used as markers for screening and identifying the transgenic components based on the nucleic acid detection technology.Here,we employed the loop-mediated isothermal amplification(LAMP)technology coupled with neutral red,an indicator dye,to establish a colorimetric detection method for maize endogenous gene and a commonly used exogenous gene.Specific primers for the endogenous gene of maize,IVR,and the exogenous genes,bar,pat,and cry1Ab were designed based on LAMP.Based on our results,the specific detection of the IVR gene,the bar gene in GM maize Bt176,and the pat gene in GM maize strains,TC1507 and 59122,could be accomplished according to the real-time LAMP fluorescence reporting system.The designed cry1Ab gene LAMP primers could specifically amplify GM maize Bt176,but not the GM maize strains,Bt11 and MON810.Nevertheless,a visual detection method was developed using 5 mmol/L Tris-HCl and 100μmol/L neutral red dye and it could be used to detect the IVR gene and the exogenous genes,bar,pat,and cry1Ab,within 1 h.Based on our findings,the visual results were consistent with those of real-time fluorescence LAMP,thereby indicating the accuracy and reliability of our visual detection method.In conclusion,the established visual LAMP method can be employed for the detection of GM maize.This method is rapid and simple,and has great potential for use as an on-site detection tool.
作者
王嘉钰
董娟
唐卓
WANG Jiayu;DONG Juan;TANG Zhuo(Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 100049,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2019年第6期1497-1502,共6页
Chinese Journal of Applied and Environmental Biology
基金
四川省青年科技创新研究团队项目（2017TD0021）
成都市科技项目（2016-HM01-00371-SF）资助~~
关键词
环介导等温扩增
转基因
基因特异性
可视化检测
现场检测
loop-mediated isothermal amplification(LAMP)
genetically modified(GM)
gene-specific
visual detection
onsite detection
