Validated method for quantification of genetically modified organisms in samples of maize flour.
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.
Kunert
Renate
R
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Renate.Kunert@boku.ac.at
Gach
Johannes S
JS
Vorauer-Uhl
Karola
K
Engel
Edwin
E
Katinger
Hermann
H
D004247:DNA / Q000032:analysis*; D005433:Flour / Q000032:analysis*; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D003313:Zea mays / Q000235:genetics*
D004247:DNA