Background and objectives

Approximately 60 different genetically modified (GM) plant lines have been officially assessed and approved world-wide. In Europe, products from ten transgenic rapeseed and maize lines have been registered as being in compliance with the Novel Foods Regulation EC 258/97. In addition, a transgenic maize species (Bt 176) and a soybean species (RoundupReady^TM) were authorised to be placed on the European market, according to Regulation EC 90/220 in 1996 and 1997, respectively. According to Article 8 of Regulation EC 258/97, foods derived from genetically modified organisms (GMOs) have to be labelled if either recombinant DNA or a new protein can be detected. Thus, highly sensitive and specific methods are needed to control compliance with the legal requirements by competent food control laboratories, but also for company quality control.

The aims of the project were firstly to develop and standardise detection methods for the identification of foods containing GMOs. Secondly, to establish DNA extraction methods for raw, processed and complex foods. The third aim was to perform ring testing (i.e. validation) of six selected detection methods, and to study possible means of enhancing efficiency of analysis and sample throughput. The final aim was to develop a database containing data on GM food world-wide.

Approach and methodology

The unambiguous identification of a transgenic element in a food product is, in most cases, easily possible if information about the modified sequences is available. Thus, the project focused on DNA analytical methods such as the polymerase chain reaction (PCR) and its experimental variants (e.g. PCR-ELISA), and direct hybridisation with specific probes. An essential prerequisite of these detection methods is the preparation of high quality DNA from complex food matrices. This was addressed by analysing sequences which are most commonly used in GM foods, so called marker genes. Immunological methods to detect heterologous proteins were also developed.
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Main findings and outcome

PCR-based methods were developed and tested for all GM foodstuffs available on the European market during the project, as well as some transgenic materials which had not been officially approved. All primer, probe and PCR conditions applied in this project for the specific detection of either a genetic alteration, or the detection of marker genes and control systems, have been listed in the Final Report and partly added to the DMIF-GEN database. This database contains useful information about commercialised GM foods (e.g. approval data, sequence inserted, DNA extraction and detection method etc.). Since the DMIF-GEN database contains no confidential data it will be freely accessible via internet. Thirty-three detailed descriptions for DNA extraction methods from raw and processed foods were submitted and gathered in a separate data collection, but were also partly added to the DMIF-GEN database. The common extraction method based on precipitation of DNA with a buffer containing CTAB (cetyl-trimethylammonium bromide, which creates a complex with nucleic acids that can be isolated after treatment with organic solvents) was useful for a wide range of applications, and 12 commercially available kits were also successfully tested. A few products caused problems during DNA extraction or subsequent PCR because, either the quantity of extracted DNA was extremely low, or results after PCR were quite inconsistent. With canned meat and fish, experiments confirmed that for more highly processed products, the limit for amplification of fragments was approximately 500 bp sized amplicons. The reliability of qualitative PCR methods was successfully tested with maize (Bt176), soybean (RoundupReady^TM), salmon and tomato (Zeneca Ltd.) in six ring trials. For the first time, two trials (maize and soybean) implied a semi-quantitative approach. Four methods have been submitted to the CEN (Comité Européen de Normalisation) working group TC/258 which was established in February 1999 to set European standards for GMO detection in foods. A ring-tested PCR-ELISA approach for screening the 35-S promoter in GM soybeans will be modified for quantitative performance. Research was carried out on multiplex PCR systems, PCR-ELISA and biosensor techniques, special gel systems for amplicon characterisation ("HA-yellow"), direct hybridisation, the NASBA (Nucleic Acid Sequence Based Amplification) approach, protein diagnostic methods, AFLP (Amplified Fragment Length Polymorphism) fingerprinting and immunological techniques. The results point to overall feasibility but also to restrictions with respect to establishing standard procedures, future development of quantitative assays, sensitivity/specificity compared with PCR-systems or applicability on highly processed materials.
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Conclusion

The project results highlight the power of DNA analysis, in particular PCR-based techniques in the rapid development of methods for GM detection in foods. In terms of recently introduced GMO threshold values, the expansion of PCR to quantitative methods becomes particularly relevant. To accelerate future development of methods and dissemination of results, the DMIF-GEN database in parallel to published single detection methods, will be at hand for broad distribution of the necessary scientific information.