抗草甘膦转基因大豆DNA标准分子构建及多重荧光PCR检测体系的建立

Construction of Reference Molecule and Multiplex Real-Time PCR for Genetically Modified Soybean Resistant to Glyphosate

[目的]将抗草甘膦转基因大豆中的CaMV35S、NOS和CP4 EPSPS 3种外源基因融合在PMD-18T载体并建立三重实时荧光PCR检测方法,实现通过一次PCR反应就能完成抗草甘膦转基因大豆的转基因成分检测。[方法]通过PCR扩增和基因克隆的方法构建含有CaMV35S、NOS和CP4 EPSPS外源基因的质粒,设计TaqMan探针建立三重荧光PCR检测体系。[结果]成功构建融合抗草甘膦转基因大豆3种外源基因质粒PMD18T/SOY,并建立三重荧光PCR检测体系,检测限值为10-3 ng/μL,线性关系良好r,2在0.998以上。[结论]构建同时含有多种外源基因的质粒作为转基因检测的阳性参照物并建立多重PCR检测体系,不仅可降低试剂成本、节省时间和人力,而且可以减少PCR反应次数和加样环节,降低PCR污染的几率。

[Objective] Plasmid PMD18T/SOY harbored exogenous gene CaMV35S,NOS and CP4 EPSPS from genetically modified soybean resistant to glyphosate was constructed.The multiplex real-time PCR method with TaqMan probes was explored and established to detect the genetically modified soybean resistant to glyphosate,which obtained the results in one tube only in a PCR reaction.[Method]The genes of CaMV35S,NOS and CP4 EPSPS were linked with PCR and cloned into Plasmid PMD18T.The detection system of genetically modified soybean using the multiplex real-time PCR with TaqMan probe was set up.[Result] The plasmid PMD18T/SOY that contained three exogenous genes of genetically modified soybean resistant to glyphosate was successfully constructed and the multiplex real-time PCR detecting system,established.The limit of quantitation was 10-3 ng/μL and the r2 above 0.998.[Conclusions]Construction of the plasmid contained exogenous genes as a positive reference template and the establishment of multiplex real-time PCR detection system not only can reduce the cost of detection reagent,time and labor,but also can save time and additional steps,thus to improve efficiency and avoid contamination.