On July 22, 2003, the Council of Ministers of the European Commission formally adopted two proposals relating to genetically modified organisms, regulating the marketing and the labeling of foodstuffs and feedingstuffs obtained from GMO. Labeling was already mandatory for the products containing more than 1% of GMO or their derivates and becomes now also necessary if the food contains more than 0,9 % of GMO. Labeling was also made compulsory for food which was not concerned before such highly refined corn and soybean oils (it is the genetic origin that prevails). On July 23, 2003, the Commission published recommendations (2003/556/EC) aiming to ensure the coexistence of genetically modified cultures and other cultures. These two regulations are a complement to directive 2001/18 on voluntary dissemination of GMO's in the environment and announce the end of the moratorium concerning the requests for introduction of new GMOs on the European market. Regulations 1829/2003/EC, 1830/2003/EC and 641/2004/EC have established a harmonized system of traceability for GMOs, they have introduced an obligatory labeling for genetically modified feed and reinforced the rules relating to the labeling of genetically modified foodstuffs. It is thus absolutely required to have new tools which make it possible to detect the plant species in a mixture, the GMOs which could be associated to it and to determine the GMO content. Moreover, the tools must be able to quickly analyze a great number of possibilities and their limits on highly processed products must be known.
Objectives
The first objective of this project consists in the development of a screening method for detection of plant species. To contribute to an effective identification of GMOs, it is indeed paramount to be able to determine which are the plant species present in the agro-food sample to be analyzed. This screening was considered by means of microarrays and Real Time PCR. The second objective is the evaluation of possibilities to quantify GMOs or their derivatives on biochips. The interest of a quantitative biochip is that it should allow a quantitative screening compared to a threshold value (for example 0.9%). For example, any sample that the biochips would quantify below a GMO content of 0.7% would certainly be considered below the threshold of 0.9% and as soon as a value of 1.1% would be exceeded, one would be assured to be above 0.9%. Concerning the remaining range going from 0.7 to 1.1%, a new measurement should be done by Real Time PCR to determine more precisely the GMO content. Real Time PCR will indeed remain more accurate due to the fact that it is not based on an endpoint detection. However, the microarray keeps the obvious advantage to be able to carry out a considerable quantity of analyses with a limited number of PCR. This feature would be reinforced within the framework of quantitative biochips.
Results obtained
For plant species detection, the first targets were based on sequences of the following genes : rbcL (chloroplastic DNA), sucrose synthase and rubisco activase. Selected sequences have some homologous sequences on which "universal" primers were designed. They surround more variable regions to distinguish species by use of capture probes (microarrays) or double dye probes (Real Time PCR). We observed that rbcL targets (high copy number targets per cell) are more sensitive than sucrose synthase or rubisco activase targets (low copy number targets per cell). The microarray approach was not enough specific with just one type of target so several targets were spotted on microarrays. These slides allow to give information on composition of several agrofood products. New genes for plant species detection were investigated and primers and probes specific to these genes were developed (flax, potato, chicory). Other specific targets were searched in the literature (wheat, cotton, sunflower, rice, tomato). Their specificity and applicability were demonstrated by Real Time PCR. The second part of the project consist in the evaluation of the possibilities of quantification on microarrays. Methods investigated link the ratio between intensity of the specific and the transgenic spots to the GMO percentage. This leads to the building of calibration curves. Three differents approaches were considerered. - A first method used certified reference flours as standards. This allowed to built a calibration curve for small GMO levels. For higher levels, some problems of spots saturation and competition between targets were met. - The second method required development of new plasmidic standards. The plasmids include 2 targets (detection of specific and transgenic targets) of which internal sequences were modified to allow a separate distinction of the set of 5 plasmids by the use of hybridation probes or capture probes. Real Time PCR shows that the standards can not be amplified together at different concentrations because of a competition between targets. Indeed standards present in smaller amounts are no more detected. Efficiency of plasmids can also differ in spite of the homologies of size, GC content and Tm. - The third technique is based on competitive PCR and requires "dual "primers. A part of the sequence of those primers is specific to the sequence to amplify. The other part, the "universal" part is specific to an another primer ("universal " or "head" primers). After solving different problems, the technique allowed to establish calibration curves for the Roundup Ready soybean. The method was also transposed for quantification of Bt11 and MOn 810 maizes. The participation to a prevalidation study of a screening GMO Microarray was also realised during the project. The study showed the suitability of the microarray to detect GMOs. Some tests performed on agro-food products enforced the conclusions of the study.
Partners
This project is the fruit of a collaboration between FUNDP (Unité de Biologie et Biochimie Cellulaire, Namur) and Department Valorisation of Agricultural Products (Unit Anthentication and Traceability) of CRA-W at Gembloux (Walloon region).
