双重实时荧光定量PCR检测转基因植物常用调控元件的适用性研究

Study on the applicability of duplex real-time fluorescence quantitative PCR to detect the common regulatory elements of genetically modified plants

中文关键词:  转基因植物  调控元件  双重  实时荧光PCR

英文关键词:genetically modified plants  regulatory elements  duplex  real-time PCR

中文摘要:

      目的 建立一种双重实时荧光定量PCR检测转基因植物中常用调控元件pCaMV35S和tNOS。方法 通过扩增多个植物DNA来测定所建立的反应系统的特异性, 将转基因棉花的模板DNA稀释5个梯度, 检测此方法检出限(limit of detection, LOD), 建立标准曲线判断该方法的定量能力, 通过相对模拟掺假试验测定方法的灵敏度。结果 特异性试验的检测结果显示此系统具备较强的特异性, 能够区分含有上述两种调控元件的转基因植物和非转基因植物。此方法中, pCaMV35S基因的检测限为1 ng, tNOS基因的检测限为10 ng。依托此方法建立的标准曲线的扩增效率分别为96%和87%, r2均大于0.99, 方法具有较好地定量能力, 相对灵敏度达到1%, 满足混合样品中转基因成分的检测要求。结论 建立的双重实时荧光PCR方法表现出较强的特异性和较高的灵敏度, 有高通量、低成本以及定量检测的能力。

英文摘要:

      Objective To develop a method for the detection of common regulatory elements pCaMV35S and tNOS in genetically modified plants by duplex real-time real-time fluorescence quantitative PCR. Methods The specificity of the system was determined by amplifying multiple plant DNA, and the template DNA of genetically modified cotton was diluted by 5 gradients to detect the limit of detection (LOD) and a standard curve was established to determine the quantitative ability of this method, the sensitivity was determined by simulated adulteration experiments. Results The results of the specificity test showed that the system had strong specificity and could distinguish genetically modified plants and non-genetically modified plants containing the two regulatory elements. The LOD of this method for pCaMV35S gene was 1 ng, and tNOS was 10 ng. The amplification efficiency of the standard curve established by this method was 96% and 87%, respectively, and r2 was greater than 0.99. The method had quantitative ability. Moreover, the relative sensitivity of the method up to 1%, which met the requirements for the detection of genetically modified ingredients in the mixed samples. Conclusion The developed duplex real-time fluorescence quantitative PCR method shows strong specificity and high sensitivity, and is capable of high throughput, low cost and quantitative detection.