转基因大豆及其制品二重荧光定量PCR的建立与验证

研究选用花椰菜花叶病毒35S启动子(P-35S)和根癌农杆菌胭脂碱合成酶基因终止子(T-NOS)为靶标基因,运用多重实时荧光PCR技术对转基因大豆及其制品进行快速筛选检测。实验通过样品核酸提取与质控,多重引物及荧光探针的设计与筛选,反应条件和反应体系的对比优化,摸索出二重荧光定量PCR检测的最佳反应体系。同时通过特异性、重复性、灵敏性和适用性实验验证,确保了该方法在同时检测2个靶标基因时,无荧光信号的相互干扰,不会出现假阴性和假阳性结果。结果表明,方法特异性高,可同时筛查两个靶标基因,扩增效率在90%~110%之间,标准曲线决定系数R²>0.98,确定了最低检测限为2拷贝/μL。开发和建立的二重荧光定量PCR检测技术可以实现一管多检的实际需要,降低试剂成本,缩短检测时间,为大豆及其深加工产品转基因成分的快速检测提供了有效方法,为促进食品进出口提供技术保障。

Establishment and verified of a duplex fluorescence quantitative PCR for screening of genetically modified soybeans and products

Duplex real-time fluorescent polymerase chain reaction technique(polymerase chain reaction,PCR)with simultaneous detection two target genes was established to screening genetically modified soybeans and deeply processed products. Multiplex primers and fluorescent probes based on the commonly used exogenous sequence of cauliflower mosaic virus 35S promoter(P-35S),Agrobacterium tumefaciens nopaline synthase terminator(T-NOS)was designed,reaction conditions and reaction systems was optimized,specificity of the duplex systems,the repeatability,sensitivity and applicability was validated to ensure two target genes could be detected with this method at the same time,with out the interference of fluorescent signals and false negative and positive results. The results indicated this method was repeatable,special and sensitive. The efficiency of amplification was between 90% and 110%,and the correlation coefficient of the standard curve R2 was greater than 0.98. The determined limits for the multiplex qPCR assays was 2 copies/μL in soybean samples. This study could be widely used screening detection of genetically modified soybeans and deeply processed products with the litter reagent cost and the shorter testing time in import and export. In turn,it would provide technical guarantee for the promotion of agricultural products and food exports.