多重RT-PCR快速检测转基因油菜

Multiplex RT-PCR for Rapid Detection of Transgenic Rape

全球转基因油菜的数量和种类日益增多,其所带来的潜在风险也备受人们的关注,迫切需要开发一套精准、高效、便捷检测转基因油菜的技术体系。本研究针对花椰菜花叶病毒35 S启动子(pCaMV 35S)、膦丝菌素乙酰转移酶(bar)、5-莽草酸-3-磷酸合成酶基因(epsps)3个目标基因的序列,以及油菜内源基因cruciferinA (CruA)序列设计四重实时荧光定量聚合酶链式反应(Multiplex real-time fluorescence quantitative,PCR)特异性强的引物和多重荧光探针。通过对多个转基因油菜材料验证,结果表明该方法检测特异性强,重复性好,4个基因的扩增效率都在90%~105%之间,标准曲线相关系数R2都大于0.99。转基因成分bar,epsps和pCaMV35S的检出限为0.1 ng/μL,内参基因CruA的检出限为0.01 ng/μL。本研究建立了一种能快速、高效、准确检测转基因油菜四重实时荧光定量PCR技术的检测体系。

The number and types of genetically modified oilseed rape are increasing in the world, and the potential risks brought by it have also attracted people’s attention. There is an urgent need to develop a set of accurate,efficient and convenient technical system for detecting genetically modified oilseed rape. This study focused on the sequence of three target genes of cauliflower mosaic virus 35 S promoter(pCaMV 35S), phosphinothricin acetyltransferase(bar), 5-shikimate-3-phosphate synthase gene(epsps), and rape. The endogenous gene cruciferinA(CruA)sequence design quadruple real-time fluorescence quantitative polymerase chain reaction(Multiplex real-time fluorescence quantitative PCR) specific primers and multiple fluorescent probes. Through the verification of multiple genetically modified rapeseed materials, the results showed that the method has strong detection specificity and good reproducibility. The amplification efficiency of the four genes is between 90% and 105%, and the correlation coefficient R2of the standard curve is greater than 0.99. The detection limit of the transgenic components bar, epsps and p CaMV35S is 0.1 ng/μL, and the detection limit of the internal reference gene CruA is 0.01 ng/μL. This study established a detection system that can quickly, efficiently and accurately detect genetically modified rapeseed quadruple real-time fluorescent quantitative PCR technology.