多重实时荧光PCR快速检测转基因大豆及其加工产品

本研究运用多重实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因大豆及其深加工制品进行筛选检测。通过设计大豆内源基因植物凝集素(Lectin)和常用的外源基因花椰菜花叶病毒35S启动子(CaMV35S)、根癌农杆菌胭脂碱合成酶基因终止(nos)的特异性引物和探针,反应条件和反应体系的优化,特异性、重复性和灵敏性的实验比对分析等开发建立了多重荧光定量PCR检测技术。以10%Roundup Ready转基因大豆标准品为材料,建立并优化转基因大豆的定量检测体系,对大豆中的转基因成分进行定量分析。结果表明:该方法重复性好,检测特异性强,扩增效率在90%～110%,标准曲线相关系数R2≥0. 98,确定了最低检测限为每20μL反应2. 4个拷贝。结论:由于使用多重实时荧光PCR技术,可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,为促进农产品和食品进出口提供技术保障。

Establishment of a Multiplex Fluorescence Quantitative PCR for Detection of Genetically Modified Soybeans and Processed Products

Multiple real-time fluorescent polymerase chain reaction technique (polymerase chain reaction, PCR) was established to screening test in the genetically modified soybeans and deeply processed products. Methods: Specific primers and Taqman probes based on soybean endogenous gene Lectin and the commonly used exogenous sequence of cauliflower mosaic virus 35S promoter (CaMV35S), Agrobacterium tumefaciens nopaline synthase terminator (nos) was designed, reaction conditions and reaction systems was optimized, and specificity of endogenous genes and exogenous gene was validated. The standard curves of endogenous reference genes and two exogenous gene were established with 10% Roundup Ready transgenic soybean and the transgenic components in soybean were quantitatively analyzed. 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 is greater than 0.98. The determined limits for the multiplex qPCR assays were 2.4 copies/reaction in soybean samples. Conclusions: This study can be widely used screening detection of genetically modified soyben and deeply processed products with the litter reagent cost and the shorter testing time in import and export.