Comparison of Plasmid and Genomic DNA Calibrants for the Quantification of Genetically Modified Ingredients

Real-time PCR is the method of choice for the quantification of the genetically modified (GM) content of food. Genomic DNA extracted from certified reference materials (CRMs) is tested for the presence of specific transgenic elements and used for calibration purposes. Recent EU Commission recommendations advocate expressing the GM content as the percentage of the number of GM target DNA sequence per target taxon specific sequence (2004/787/EC). CRMs currently available are certified gravimetrically on their mass percentage but not for copy numbers of transgenic and endogenous targets. Reference materials certified for copy numbers are therefore desirable to fulfil this EU recommendation. An inter-laboratory trial was conducted to evaluate the potential for using plasmid DNA compared to genomic DNA for quantitative PCR calibration in terms of copy numbers, using the model system of Roundup ReadyTM soya. Data was analysed from 48 randomised real-time quantitative PCR plates under conditions of reproducibility across three international laboratories. Four experiments were conducted using a combination of plasmid and genomic templates both as calibrants and sample unknowns. Results demonstrated that the specific plasmid DNA used in the current study provided a suitable alternative to genomic DNA for use as a calibrant in GM quantitation. In the current investigation, plasmid calibrants gave equal or better performance characteristics in terms of closeness to the expected value and precision, than their genomic equivalents.JRC.D.2-Reference material