Project summary
of the GMOchips-project
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EU-Commission Reserach
Project Measurement
& Testing, Contract No. G6RD-CT2000-00419. Duration: 2001-2004 Project summaryOver the last years, there has been a dramatic and continuing increase of the surface planted with transgenic crops. The five principal transgenic foodstuffs are maize, soybean, rapeseed / canola, tomato and potato. The European Union informs the consumers of the presence of a trangenic foodstuff by labelling of the no more "substantially equivalent" ingredients (Food Regulation 258/97 and the Council regulations 1139/98, 49/2000 and 50/2000). The fortuitous presence of recombinant DNA or modified protein above of a defined threshold of 1 % of ingredients in the foods will lead to unambiguous food labelling. The development and application of reliable and quantitative analytical detection methods are thus essential in order to perform and to control food labelling as well as for the possible development of “GMO free” production schemes and to control plant importation at the EU border. The project is a continuation and extension of 2 other European research projects (shared cost RTD Project QLK1-CT-1999-01301 "qpcrgmofood" and the DMIF-GEN SMT4-T96-2072):
The microarray test is adaptable to the increasing emergence of new GMOs in the food and feed markets. Technical and IPR reasons require the development of new quantitative PCR (QPCR) based detection methods alternative to the "real-time" QPCR proprietary techniques. The microarray approach should permit to suspect the presence of unidentified GMO in a sample, provided its pattern of hybridisation on micro-array differs of the ones stored in a database of the approved GMO (e.g. P35S + CryIA(b) gene + Tnos + other DNA targets). The proposed biochips combine the advantages of the unambiguous identification and a multiparametric approach to furthermore detect unexpected GMO via their specific patterns. The use of micro-arrays allows a test miniaturisation and a simultaneous detection of many DNA sequences while the multiplex real-time QPCR will be definitely limited (i) by the sequences homologies between primers and probes and (ii) by the technical difficulties to quantitatively distinguish more than 3 fluorophores (fluorescent reporters). The proposed technology, low-density chips, and the reading device used are user-friendly, handy and cheap. The low density biochips technology (lower than 1000 spots/cm2) with colorimetric detection is a non proprietary technology which can be transferred in less equipped laboratories and in developing countries. The plants concerned by this project are: beet, chicory, corn, cotton, flax, potato, rapeseed, rice, soybean, squash and tomato. The work will be in a first time focused on plants and their corresponding GMO not already taken into account by the previous research projects dedicated to the QPCR on the edge fragments of the inserts. It will particularly focus on the crops no longer regulated in the USA but not yet approved in the EU. Most of the biotech companies regulate their GMO first on the North American continent. Below we present a brief description of the work performed by each of the 7 partners: FUNDP co-ordinates the programme and will be more particularly in charge of the development of the low density biochips, the optimisation of the capture probes and the quantification of the detection using internal standard. INRA will co-ordinate the optimisation of the PCR test, which should incorporate the d-UTP-biotin nucleotides (WP-2). This partner will also develop the PCR tests on the plant specific sequences (endogenous genes), necessary to determine the 100% of ingredient of sugar-beet and potato. This partner will then focus its work more particularly on two herbicide tolerances (phosphinothricin, glyphosate). The corresponding sequences will be cloned as well as the sequences useful for competitive PCR and the primers designed for consensus PCR. The quantification limit of these consensus PCR will be determined on several GMO. TEPRAL will take part in the ring-test, which will consist in the final step of the project. LGC’s role will be the optimisation of hybridisation conditions for target and capture probes in various GMO. This will make use of the Lightcycler to determine actual Tms using melting curve analysis. Tm data will then be tested with the biochip detection system at this stage to determine optimal hybridisation conditions. The partner's experience with optimising hybridisation conditions using cosolvents will also be utilised. Moreover, this partner will take part in the ring-test. DGCCR will develop the PCR tests on the plant specific sequences (endogenous genes) necessary to determine the 100% of ingredient of rice. Then, he will work more especially on two herbicide tolerances (Sulfonylurea and Bromoxynil). The corresponding sequences will be cloned as well as the sequences usefull for competitive PCR and the primers designed for consensus PCR. Their quantification limit will be determined on several GMO. DGCCR will also take part to the ring-test. CSIC will concentrate on species on which their laboratory has long experience on Molecular Biology studies such as corn, rice, tomato, potato, which are included on the list of proposed GMOs to be studied within the project. They will work in the design of the primers required for the amplification of the sequences that are going to be used in the construction of the plasmid bank specific to this GMO and in the design of the consensus primers. This partner will also take part in the biochips development and in the ring-test. BATS will collect the identified internal sequences of the inserts of the GMO approved or not in the EU database. They already coordinates such data based publicly accessible on the web. AAT, the subcontractor, will be in charge of the production on a large scale with quality control, necessary for providing the biochips for the ring-test. More information about the GMOchips-project:
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