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|Title:||Towards Metrological Traceability for DNA Fragment Ratios in GM Quantification 1. Effect of DNA Extraction Methods on the Quantitative Determination of Bt176 Corn by Real-Time PCR|
|Authors:||CORBISIER PHILIPPE; BROOTHAERTS WIM; GIORIA SABRINA; SCHIMMEL HEINZ; BURNS M.; BAOUTINA Anna; EMSLIE K.; FURUI S.; KUROSAWA Yasunori; HOLDEN M.; KIM H.-H.; LEE Yun-Mi; KAWAHARASAKI M.; SIN D.; WANG J.|
|Citation:||JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY vol. 55 p. 3249-3257|
|Publisher:||AMER CHEMICAL SOC|
|Type:||Articles in periodicals and books|
|Abstract:||An international CCQM-P60 pilot study involving 8 national metrological institutes was organised to investigate if the quantification of GM maize powder by real-time PCR was affected by the DNA extraction methods applied. Four commonly used extraction methods were compared for the extraction of DNA from a corn Bt176 maize powder. Extracts were assessed for DNA yield using a fluorescent-based DNA quantification assay and for quality measuring their absorbance ratios at 260 nm / 280 nm and 260 nm / 230 nm. The CTAB-based method yielded the highest quantity and DNA template quality. A difference in the 260 nm / 230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of endogenous genes zein and hmg as well as trangenic sequences covering th cryIAb gene and a plant-Bt176 junction fragment were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. An underestimation of the GM percentage was observed by all laboratories for the highest concentration of DNA template suggesting an inhibition in the amplification of the GM targets. That inhibition could be avoided by additional clean up of the DNA template. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied. The relative measurement uncertainty under repeatability conditions was lower than 25 % and represents most probably the best level which can be presently achieved for such amplification technologies.|
|JRC Directorate:||Health, Consumers and Reference Materials|
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