standardised, specific, quantitative detection of genetically modified food
Available methods for the detection of genetically modified (GM) materials
can only be used to screen for the presence of potential GM material,
or to detect and identify known GM material. Consequently, a potential
danger arises from the failure to detect non-approved GM organisms (GMOs).
GMO approval covers a specific genetic modification (product of a transformation).
Because this can be used in more than one GMO, such elements are not reliable
identifiers of specific transformations. Clearly, there is a regulatory
need for detection methods which can distinguish between approved and
non-approved GMOs and quantify GMO content. Thus detection methods specific
for each transformation event (using the junction between the modified
DNA and the part of the host genome where the modified DNA is integrated)
are necessary. Very few plant, species-specific genes which can be used
as a reference for quantification of GMO content, have been identified.
A qualitative analysis that can detect and identify more than a single
GMO in a single reaction, has clear cost-efficiency potential. Furthermore,
molecular methods which can influence the sensitivity and reliability
of polymerase chain reaction (PCR)-based detection assays must be optimised.
Finally, validation of these methods is critical to assess their reliability.
There is also an urgent and growing need for European and international
standards for GMO detection.
aims of this project were, firstly, to develop reliable and transformation
event specific tests for qualitative and quantitative detection of genetic
modifications in food, for at least 12 GMOs. Secondly, to develop reliable
and transformation event specific multiplex tests for determination of
the diversity of genetic modifications in food. Finally, to investigate
how improved methods for detection of genetically modified foods will
influence consumer confidence in food security, science and risk regulators.
This project comprises six work packages. The first of these involves
the identification of both the application and the limitations of a standard
DNA extraction protocol. This will be done by examining the effects of
various modifications of the extraction protocol on a variety of matrixes.
PCR quantification is usually relative to a single-copy gene specific
for the GM host species. The second work package will identify and characterise
suitable species-specific reference genes, and develop reference gene-specific,
primer/probe sets for qualitative and quantitative PCR amplification and
detection. This work will involve screening the literature and DNA sequence
databases, sequencing uncharacterised candidate genes, and also empirically
testing putatively suitable primers and probes. Copy number per cell will
be examined by standard DNA hybridisation techniques. The third work package
involves sequence characterisation of transformation events. Sequence
data will be requested from biotechnology companies or other sources on
a collaborative basis. If necessary, appropriate pure DNA fragments containing
the junction regions, will be isolated and sequenced. In the fourth work
package, transformation event specific, primer/probe sets will be developed
and tested. Specific PCR primers and detection probes will be developed,
and as primers and probes may perform differently in different systems
and assays, several alternative primers and probes targeted for the detection
of each GMO are needed. Thus, techniques will be developed for the qualitative
detection of GMOs in single primer-probe assays, in multiplex primer-probe
assays, as well as quantitative detection assays for GMOs. In the fifth
work package, techniques used in ring-trials will be validated. The final
work package examines the socio-economic impact of GMO regulation and
detection. A questionnaire survey in the United Kingdom, Norway and Italy
will provide information about the impact of tests which improve traceability
of genetically modified foods on consumer confidence.
to view graphic (pdf)
Main findings and outcome
DNA molecular techniques are being established and will be tested on beer,
sweet and salted biscuits, glucose syrups, lecithins, starches, oils,
and proteins. Candidate reference genes for maize and soya, including
the well known soya lectin, maize invertase, zein and high-mobility-group
(MHMG) genes are being examined and characterised. The junction between
the host genome and inserted DNA is being characterised for RoundupReadyTM
soya, Bt176, Bt11, Mon810 and T25 maize. Available published primers and
probes as well as new and unpublished primers and probes are being tested
and evaluated, to prepare the development of multiplex detection systems
and examine the effect of various parameters on the accuracy of quantifications.
Validation of detection methods in ring trials will be performed with
pure DNA samples, and a questionnaire survey will be carried out.
This project should provide information and methods essential for the
detection of GMOs, and to identify the impact of this improved detection
on consumer confidence.
Lipp M., Anklam E., Brodmann P., Pietsch K., Pauwels J., "Results
of an interlaboratory assessment of a screening method of genetically
modified organisms in soybeans and maize".
Food Control, 10, 1999, pp. 379-384.
M., Pijnenburg H., Gendre F., Brignon P., "Real-time quantitative
PCR detection of genetically modified maximizer maize and RoundupReadyWTM
soybean in some representative foods".
Journal of Agricultural and Food Chemistry, 47, 1999,
Wurz A., Bluth A., Zeltz P., Pfeifer C., Willmund R., "Quantitative
analysis of genetically modified organisms (GMO) in processed food
by PCR-based methods".
Food Control, 10, 1999, pp. 385-390.
February 2000 - January 2003
National Veterinary Institute
Norwegian Food Research Institute
M. De Loose
Agricultural Research Centre
G. van den Eede
Joint Research Center
Unilever research laboratory
DG de la concurrence, de la consommation et de la répression
Institute of Food Research
Instituto de Biología Molecular de Barcelona (ES)