of methods to identify foods produced by means of genetic engineering
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 (RoundupReadyTM)
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.
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
(RoundupReadyTM), 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.
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.
Hupfer C., Hotzel H., Sachse K., Engel K.-H., "Detection of
the genetic modification in heat treated products from Bt maize
by polymerase chain reaction".
Z. Lebensm. Untersuch. Forsch. A, 205, 1997, pp. 442-445.
Straub J.A., Hertel C., Hammes W.P., "A 23S rDNA-targeted polymerase
chain reaction-based system for detection of Staphylococcus aureus
in meat starter cultures and dairy products".
J. Food Prot., 62 (10), 1999, pp. 1150-1156.
Vollenhofer S., Burg K., Schmidt J. and Kroath H., "Genetically
modified organisms in food-screening and specific detection by polymerase
J. Agric. and Food Chem., 47 (12), 1999, pp. 5038-5043.
Zagon J., Broll H., Schreiber G.A. and Schauzu M. (eds.), Proceedings
of the European Research Project SMT4-CT96-2072, BgVV-Hefte
05/1999, Bundesinstitut für gesundheitlichen Verbraucherschutz
und Veterinärmedizin, Berlin (ISBN 3-931675-07-6), 1999.
Zimmermann A., Lüthy J. and Pauli U., Z. Lebensm. Unters.
Forsch., 207, 1998, pp. 81-90.
October 1996 - October 1999
Federal Institute for Health Protection of Consumers and Veterinary
Institut für Chemo- und Biosensorik e.V. (ICB)
M.J.T. Carrondo, T. Crespo
Instituto de Biologia Experimental e Tecnologica (IBET)
Ministry of Food Agric. & Fisheries
Landesveterinär- und Lebensmitteluntersuchungsamt
H. Gaugitsch, A. Heissenberger
Universität Hohenheim (UHOH)
Bundesanstalt für Lebensmitteluntersuchung
Stockholm University (SE)
O. Käppeli, J. Lindenmeyer
University of Athens (GR)
M. Leonardi, E. Benvenuto
Istituto Superiore di Sanità
Bundesamt für Gesundheit
H. Pyysalo, E. Eklund
Finnish Customs Laboratory
H. Rüggeberg, G. Meyer
Hanse Analytik GmbH Hanse Analytik Labor
BgVV FG 425
Laboratory of the Government Chemist/Analytical Molecular Biology
Federal Biological Research Centre for Agriculture and Forestry
I. Huyghebaert, K. Messens
Universiteit Gent (BE)
A. van Hoef, H.A. Kuiper
State Institute for Quality Control of Agricultural Products (RIKILT)
Austrian Research Centre (ÖFS)