Ontwikkeling en implementatie van strategieën voor GGO kwantificering in een wijzigende Europese context: Ontwikkeling en implementatie van strategieën voor GGO kwantificering in een wijzigende Europese context

    Onderzoeksoutput: ScriptieDoctoraatsscriptie - Doctoraatsscriptie

    Uittreksel

    The recombinant DNA technology or ‘modern biotechnology’ originates from the seventies.
    After the ‘red biotechnology’ or the applications of biotechnology in the medical sector, the
    ‘green biotechnology’ started to progress. The first genetically modified plants, resistant to
    viruses and insects, were field tested in 1985. A fast growth of the number of transgenic crops
    and products on the American and European markets followed. Major advances evolved as a
    result of new insights and developments in the domain of plant transformation technologies.
    At the same time, in the European Union, the regulatory framework concerning the
    cultivation, the introduction into the environment, and the commercialization of genetically
    modified organisms (GMOs) changed rapidly. As a consequence, there was an emerging need
    for reliable methods for the detection and quantification of plant transgenic material. This not
    only in plants and seeds, but also in grains and all derived food and feed products, and thus
    covering the whole production and distribution chain.
    The research on the development and implementation of quantitative strategies of GMO
    analysis, described in this dissertation, started with an extensive analysis of the ‘GMO
    problem’ in all its facets. GMO bioanalysis in the laboratory may be called complex, in the
    first place due to the complex nature of a GMO as such. Related to this is the variation in
    genetic and structural composition, as well as in matrices which may contain or be derived
    from GMOs. Moreover, the DNA-based PCR technique is unique and calls for an adapted
    approach. When scrutinizing the complexity of GMO analysis, special focus is on the aspect
    of quantification of DNA sequences, and the expression of a relative GMO content.
    Furthermore, the unclear and incomplete EU legislation from the nineties has hampered the
    establishment of a straightforward and simple strategy of analysis.
    This research was carried out midst this restless period, which was characterized by
    evolutions. In spite of those difficulties and the typical character of GMOs as well as their
    bioanalytical detection techniques, we elaborated a system which allows to quantify GMOs in
    a precise and correct way. For this purpose, a new type of DNA standards was developed,
    which is based on cloned DNA sequences. In combination with a set of event-specific primers
    and a fluorescent TaqMan probe, the plasmid calibrators were used in real-time PCR.
    Different methods for relative GMO quantification have been experimented, and the new
    plasmid markers tested and compared to other types of more commonly used, genomic DNA
    standards. Such a quantitative system was established in this work for about ten
    commercialized GM-events. In this way, we enable a consistent implementation of EU
    regulations as to the mandatory labelling of GM-derived products. Thanks to the initiation of
    iv
    an official database of plasmid DNA standards and PCR methods for GMOs, we contribute
    towards international harmonization of GMO strategies of analysis.
    It is beyond doubt that results of GMO analyses have a high socio-economic impact. If a
    sample is positive with regard to the presence of GMOs, and if the GM-content is above 0.9
    % on the level of single ingredients, this product must be labelled. Since analytical results
    form the basis of decision making, per definition, they must always be accompanied by a
    statement of uncertainty. Measurement uncertainty, together with traceability to well-stated
    references or primary standards, are crucial elements for comparability between measurement
    results. In this thesis, we established a quality monitoring system for the newly developed
    strategy of analysis. Reliable and traceable analytical results, including their known
    measurement uncertainty, are reliant on method validation, application of internal and
    external quality control measures and compliance to the principles of ISO/IEC 17025. The
    establishment of a quality system was urged by the lack of harmonized and guiding norms or
    recommendations, and of a clear regulatory framework on the validation and quality
    assurance of GMO methods of analysis.
    With the development of methodologies for GMO detection and quantification, we principally
    aim at answering the client’s questions. Different actors in the chain, from seed supplier to
    processor, manufacturer and retailer, have their specific needs. The presented, multipurpose
    strategy for GMO analysis is highly flexible and can be applied on a routine scale. A plan
    which is based on decision trees, permits to screen for GMOs, to specifically identify a
    number of GM events, and to accurately quantify GMOs on the level of ingredients. What’s
    more, anchor PCR is put forward as a helpful tool for in-depth analysis of ‘special’ samples.
    The latter may have to do with the presence of irregularities or unknown elements in a
    sample. Anchor PCR is the only technique by which at least an indication of the presence of
    non-authorized GMOs in a sample can be obtained. Given the expected fast growth of
    transgenic area worldwide, in addition to the number of GMO products on the markets, such
    an integrated approach for GMO analysis on a large scale is more than wished.
    TaalEngels
    Plaats van publicatieGent
    Uitgever
    Gedrukte ISBN's90-5989-049-3
    Elektronische ISBN's90-5989-049-3
    StatusGepubliceerd - 1-mrt-2005

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