Validation of real-time PCR methods for the quantification of transgenic contaminations in rape seed

At present genetically modified oilseed rape (Brassica napus) is not allowed to be cultivated in the countries of the European community. This is because rape seed has to be free of any transgenic material if it is destined for growth in the European Community. However, a new regulation is forthcoming that will distinguish seed to be labeled from seed that is not to be labeled by a legal threshold value for the content of transgenic material. In this paper real-time PCR methods are described that are applicable for the quantification of transgenic contaminants after screening and identification analysis. The validation of their quantification is demonstrated for contaminants with resistance to the herbicides Basta and Roundup Ready in samples of conventional rape seed. The limits of quantification were determined for both systems for 50 copies of the transgenic DNA in the reaction assay (confidence interval lower than 30% at a 95% probability level) corresponding to 0.1% of transgenic DNA in the total amount of genomic DNA. Results show that the real-time PCRs established are applicable with the GeneAmp sequence detection system (Applied Biosystems) as well as with the Light Cycler (Roche). The methods described in this paper can be used for the assessment of a contamination in rape seed according to future threshold regulations.