抗除草剂转基因作物实时荧光定量PC检测

建立了一种以SYBR Green Ⅰ为结合染料、快速准确检测转抗除草剂基因成分的实时荧光定量PCR方法.以转基因大豆与转基因玉米标准品为材料,通过使用特异性引物和SYBR Green Ⅰ结合染料实时荧光定量PCR技术,对转基因农作物中外源抗除草剂基因进行了定量检测,绘制了两种基因扩增的标准曲线图,根据标准曲线方程计算外源基因含量;并作了溶解曲线、检测方法检测灵敏度和精密度的分析.研究发现,两者标准曲线方程线性关系良好.R~2 值分别达到0.993 9与0.992 4.通过已知标准品进行验证,实测值与真值接近,与实际含量的相埘偏差是6.52%和7.90%.结果表明,SYBR Green Ⅰ结合染料法完全可以用于转基因农作物定量PCR检测.图5表2参11

Detection of Genetically Modified Herbicide-tolerant Crops by Real-time Fluorescent Quantitative PCR Assay

A rapid and sensitive SYBR Green Ⅰ-based real-time PCR method was developed for quantitative detection of CP4-EPSPS and PAT genes from the genetically modified (GM) herbicide-tolerant crops. The target genes from transgenic soybean and maize references were amplified to make standard curves. The transgenic ratios were then calculated according to the standard Ct-copies linear graphs of these two genes. The reproducibility and melting curves of the genes were also analyzed. The results showed that the standard equations of CP4-EPSPS and PAT genes had higher R~2 values of 0.993 9 and 0.992 4, respectively. The difference in transgenic ratios between the known standards and measured crops was only 6.52%～7.90%. These results suggest that the real-time PCR assay with SYBR Green I dye is suitable for detecting the ratios of GM crops and their derivates. Fig 5, Tab 2, Ref 11