Kunert Renate, Gach Johannes S, Vorauer-Uhl Karola, Engel Edwin, Katinger Hermann
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
J Agric Food Chem. 2006 Feb 8;54(3):678-81. doi: 10.1021/jf052257s.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.
对植物材料中痕量生物技术衍生DNA进行灵敏且准确的检测，是检测食品及其原材料中1%或0.5%转基因成分的前提条件。与酶联免疫吸附测定法（ELISA）检测表达蛋白相比，实时荧光定量聚合酶链反应（RT-PCR）扩增的样品制备更简便，检测限更低。在不同的DNA制备方法中，选择了十六烷基三甲基溴化铵（CTAB）法，该方法对起始材料具有高度灵活性，且能产生具有相关质量的足够DNA。先前采用SYBR Green检测法生成的RT-PCR数据表明，该方法对影响结果解读的样品基质和基因组DNA含量高度敏感。因此，本文描述了一种基于TaqMan探针法的实时DNA定量方法，该方法具有高准确性和灵敏度，检测限低于每个样品18个拷贝，适用于并可与高度纯化的质粒标准品以及基因组DNA样品的复杂基质相媲美。使用ValiData对结果进行了方差同质性、线性、标准曲线准确性和标准差的评估。
