Czech J. Food Sci., 2012, 30(3):247-257 | DOI: 10.17221/317/2011-CJFS

Development and verification of PCR based assay to dectect and quantify garden pea lec gene

Aleš VRÁBLÍK1,2, 3, Jan HODEK1, Josef SOUKUP2, Kateřina DEMNEROVÁ3, Jaroslava OVESNÁ1
1 Department of Molecular Biology, Crop Research Institute, Prague, Czech Republic
2 Department of Agroecology and Biometeorology, Czech University of Life Sciences Prague, Prague, Czech Republic
3 Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Prague, Czech Republic$2

Genetically modified organisms (GMOs) entering the food chain have became its part, which is necessary to monitor. GMO analyses are used as a control mechanism according to valid acquis communautaire for traceability and labeling of GMOs. Generally, approved PCR based protocols are used and they require stepwise procedures that use amplification of species specific gene as initial point. This study aims to develop and verify PCR based assay for amplification of garden pea lectin gene (Pisum sativum L.) as reference one. Lectin gene was analysed in silico, selected region was amplified and sequenced and new set of species specific primers for identification of garden pea was designed. Conditions of conventional PCR as well as real-time PCR were optimised and specificity of new primer set on DNA extracted from garden pea cultivars as well as DNA extracted from other selected species from Fabaceae family was tested. Quantification of garden pea lectin gene using real-time PCR based on SYBR Green I was optimised and performance characteristics recorded. The characteristics fit to method acceptance criteria range. Plasmid with garden pea lectin sequence was developed and plasmid is available as a positive control.

Keywords: GMO; lectin; PCR detection; real-time PCR

Published: June 30, 2012  Show citation

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VRÁBLÍK A, HODEK J, SOUKUP J, DEMNEROVÁ K, OVESNÁ J. Development and verification of PCR based assay to dectect and quantify garden pea lec gene. Czech J. Food Sci. 2012;30(3):247-257. doi: 10.17221/317/2011-CJFS.
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