双重荧光定量PCR杂交探针标记法快速鉴定转基因玉米MON89034  

Rapid Identification of Transgenic Maize MON89034 by Double Fluorescence Quantitative PCR Hybridization Probe Labeling

作　　者：王凤军 陈强 许海锋 杨伊平 周叶熹 Wang Fengjun;Chen Qiang;Xu Haifeng;Yang Yiping;Zhou Yexi(Department of Applied Engineering,Zhejiang Institute of Economic and Trade,Hangzhou,310018)

机构地区：[1]浙江经贸职业技术学院应用工程学院,杭州310018

出　　处：《分子植物育种》2023年第10期3341-3348,共8页Molecular Plant Breeding

基　　金：浙江经贸职业技术学院省属高校基本科研业务费项目(20SBYB03);浙江省基础公益研究计划项目(LGC19C-200007);浙江省大学生科技创新活动计划暨新苗人才计划项目(2021R444003);浙江省供销社课题研究项目(21SSY13)共同资助。

摘　　要：本研究创建一种简单快捷、成本低廉、灵敏准确及可靠性强的高通量鉴定转基因玉米MON89034转化体的快速检测方法。采用多重实时荧光聚合酶链反应(polymerase chain reaction,PCR)对转基因玉米MON89034品系进行检测,并针对转基因玉米MON89034靶标基因设计高效稳定的引物和探针,使用zSSIIb基因作为内源基因,避免检测出现假阴性,将反应体系及条件优化后,用实时荧光定量PCR仪进行扩增,初步建立转基因玉米MON89034双重实时荧光PCR检测方法,通过对该方法进行特异性和大样本可靠性测试验证,继而创建出一种高通量鉴定转基因玉米MON89034转化体的检测方法。通过建立的双重荧光定量PCR方法对转基因玉米、非转基因玉米和转基因大豆的DNA混合模板检测得出的结果表明,多种作物DNA混合模板可在单根PCR反应管内实现对zSSIIb基因和MON89034的同时检测,并在2 h内完成检测得到准确结果。目前,建立的该双重实时荧光PCR方法可用于多个转基因作物样品间快速筛查,检测快速,结果准确,可满足大多数检测机构日常检测的需求。In this study,a simple,rapid,low-cost,sensitive,accurate and reliable method for high-throughput identification of transgenic maize MON89034 transformants was established.Multiple real-time fluorescent polymerase chain reaction(PCR)was used to detect the transgenic maize MON89034 strain,and efficient and stable primers and probes were designed for the target gene of transgenic maize MON89034.zSSIIb gene was used as the endogenous gene to avoid false negative.After optimizing the reaction system and conditions,real-time fluorescent quantitative PCR was used for amplification,A dual real-time fluorescent PCR detection method for transgenic maize MON89034 was preliminarily established.Through the test and verification of the specificity and large sample reliability of the method,a high-throughput identification of transgenic maize MON89034 transformants was created.The DNA mixed templates of transgenic maize,non transgenic maize and transgenic soybean were detected by the established double fluorescence quantitative PCR method.The results showed that the DNA mixed templates of various crops could detect zSSIIb gene and MON89034 simultaneously in a single PCR reaction tube,and the detection was completed within 2 hours to obtain accurate results.At present,the dual real-time fluorescent PCR method can be used for rapid screening among a variety of transgenic crop samples,with rapid detection and accurate results,and can meet the daily detection needs of most detection institutions.

关 键 词：转基因 MON89034 实时荧光PCR 

分 类 号：S513[农业科学—作物学]