Trends in Food Science & Technology 17 (2006) 490–497
Review
Quantification
of DNA from
genetically modified
organisms in
composite and
processed foods
Karl-Heinz Engela*,
Francisco Moreanoab,
Alexandra Ehlerta and
Ulrich Buschb
a
&
Center of Food and Life Sciences, Chair of General
Food Technology, Technical University of Munich,
Am Forum 2, 85350 Freising-Weihenstephan,
Germany (Tel.: C49 8161 714249; fax: C49 8161
714259; e-mail: [email protected])
b
Bavarian Health and Food Safety Authority LGL,
Veterinärstr. 2, 85764 Oberschleißheim, Germany
Challenges arising from the task to quantify DNA from
genetically modified organisms (GMO) in composite and
processed foods are reviewed. Examples for distortion of the
quantification of GMO due to differences in particle size
compositions and heat-induced DNA degradation are
discussed. Ligation-dependent probe amplification is
presented as a novel approach to tackle the issue of the
increasing number of GMO to be screened and quantified.
The use of hybrid molecules as easily accessible synthetic
DNA-standards for quantitative screening of GMO via
real-time PCR is described.
* Corresponding author.
0924-2244/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2006.04.008
Introduction
The use of GMO and GMO-derived products as foods or
food ingredients is subject to regulatory oversight in a
number of countries (ILSI Europe, 2001). In the European
Union, a uniform traceability system defining provisions for
the documentation of the flow of GMO-derived products in
the food chain has been established (EC, 2003a,b).
According to the present legislation, the requirement for
labeling is no longer dependent on positive testing of
products for the presence of GMO-derived material (DNA
or protein). Consequently, even highly processed GMOderived products are covered by the new legislation,
irrespective of the detection of recombinant DNA or
proteins. Nevertheless, development and validation of
quantitative methods for GMO analysis in foods remain
essential, because the need for labeling has been linked to
certain thresholds (EC, 2003a,b).
Official methods for the event-specific detection and
quantification of material derived from authorized
transgenic crops are provided by the European Commission’s Reference Laboratory. Although these methods
represent valuable tools, the experience gained from
surveillance testing of GMO shows that laboratories have
to cope with major analytical challenges. This review
will focus on latest developments and future perspectives
of the following issues: (i) quantification of GMO in
composite foods, (ii) GMO analysis of processed
products, (iii) the challenge arising from the increasing
number of GMO to be screened, and (iv) the lack of
reference materials.
Quantification of GMO in composite foods
Industrially produced foods are usually composed of
various ingredients. Different ingredients derived from
the same GM crop may possess different properties. For
example, mixtures of corn milling fractions with different
particle size distributions are used in industrial applications (bakery or extrusion products) to regulate
important functional and sensory characteristics. Appropriate particle size distributions of the meal and flour
mixtures add important functional characteristics to the
products and are generally obtained by recombining the
previously sieved fractions (Alexander, 1987; Moser,
Kniel, Schmitz-Winnenthal, Hupfer, & Engel, 1999;
Rooney & Serena-Saldivar, 1987). Isolation of DNA
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
Fig. 1. Quantification results of corn flour/grits mixes. MeansG
CI(p!0.05). The marked lines indicate the adjusted GMO content
of 1%. (Moreano et al., 2005a).
from commonly used corn milling fractions (coarse grits,
regular grits, cornmeal and flour) revealed a strong
correlation between the degree of comminution of the
milling fractions and the DNA yields in the extracts
(Moreano, Busch, & Engel, 2005a). Mixtures prepared by
different combinations of coarse grits and flour from
conventional or transgenic corn (all adjusted to a GMO
content of 1%) were quantified using a commercially
available real-time PCR quantification kit. Accurate
quantification of the adjusted GMO content was only
possible in mixtures containing conventional and transgenic material in form of analogous milling fractions.
Mixtures of fractions exhibiting different particle size
distributions delivered significantly over- and underestimated GMO contents depending on their compositions (Fig. 1).
These data demonstrate that relative GMO quantifications may be significantly distorted if the analyzed food
contains fractions with different particle size distributions.
Especially, if the presence of GMO is limited to one of the
fractions, proportions of DNA extracted from both fractions
would not reflect the actual weight proportions of GM
material contained in the food sample.
Quantification of DNA in processed foods
Food manufacturing involves a number of processing
steps that may negatively affect the performance of
491
GMO detection methods. Fragmentation of DNA may
be initiated by grinding and milling owing to shear
forces and hydrolysis by nucleases. It is primarily linked
to processes carried out at low pH and increases
dramatically if these are performed in combination
with thermal treatment (Jonas et al., 2001; Klein,
Altenbuchner, & Mattes, 1998). Effects of DNA
degradation on the results of PCR-based detection
systems have been investigated by following diverse
manufacturing practices. It was shown that the lengths
of the selected target sequences are crucial parameters
for the detectability of DNA (Bauer, Weller, Hammes,
& Hertel, 2003; Hupfer, Hotzel, Sachse, & Engel, 1998;
Hupfer, Mayer, Hotzel, Sachse, & Engel, 1999; Straub,
Hertel, & Hammes, 1999). Qualitative assays demonstrated that longer target sequences (O300 bp) become
increasingly susceptible to degradation under stress
conditions, resulting in false negative results when
analyzing samples of processed foods or feeds.
Quantitative analysis via competitive PCR confirmed
the significant decreases in the recoveries of target
sequences in the course of processing (Hupfer, Hotzel,
Sachse, Moreano, & Engel, 2000; Klein et al., 1998).
Degradation of DNA was shown to affect limits of
detection as well as limits of quantification (Spiegelhalter, Lauter, & Russell, 2001). For quantitative
approaches the importance to use plant-specific reference
genes that allow the normalization of quantification
results was emphasized (Hübner, Studer, & Lüthy,
1999). Due to the improved stability of shorter DNA
sequences towards stress conditions there is a commonly
acknowledged approach to aim at target sequences
%200 bp in qualitative and quantitative assays. These
assays were generally considered as appropriate for the
analysis of processed products, as it was assumed that
recombinant and reference target sequences possessing
approximately the same length will be equally degraded
in the course of processing (Wiseman, 2002).
Currently used methods for the relative quantification
of recombinant DNA are based on real-time PCR
(Anklam, Gadani, Heinze, Pijnenburg, & Van den
Eede, 2002; Miraglia et al., 2004). Development and
validation are generally accomplished by analyzing
certified reference materials (Institute of Reference
Materials and Measurements, Belgium), which represent
mixtures of flours containing defined proportions of
GMO-derived material. Following accuracy testing,
quantitative assays have been directly applied to the
analysis of processed foods. Experiments demonstrating
that neither food composition nor processing influence
the trueness of relative GMO quantification have not
been performed (Höhne, Santisi, & Meyer, 2002; Holck,
Va, Didierjean, & Rudi, 2002; Pietsch & Waiblinger,
2001; Vaitilingom, Pijnenburg, Gendre, & Brignon,
1999; Wurz, Bluth, Zelz, Pfeifer, & Willmund, 1999).
Some studies focused on the analysis of processed
492
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
Fig. 2. Effect of thermally induced DNA degradation on the recovery of GMO contents after quantification of coarse corn grits via Methods A
and B. Error bars indicate standard deviations of the means. (Moreano et al., 2005a).
samples with known GMO proportions, but did not
determine the GMO proportions of the unprocessed
counterparts (Berdal & Holst-Jensen, 2001; Terry &
Harris, 2001; Taverniers, Windels, Van Bockstaele, &
De Loose, 2001). It remained unclear whether the found
discrepancies in quantification results were consequences
of DNA degradation or whether they are to be
considered as inherent bias of the quantification system.
Results of an international ring trial regarding the
validation of a method for the quantification of
transgenic maize (line Bt176) allowed an accurate
quantification of GMO contents in unprocessed reference
materials, but showed a significant underestimation of
GMO contents in heat-sterilized samples (Broll, Zagon,
Butschke, & Grohmann, 2002; ISO/DIS, 2003). The fact
that the transgenic-specific target sequence (129 bp) was
longer than that used for the detection of the reference
gene (79 bp) permitted the assumption that distortions in
the results of relative quantification could result from an
unequal sensitivity of target sequences towards processing parameters.
Recent studies assessed to what extent minor
differences in the lengths of the targeted sequences of
recombinant and reference genes occurring in these
methods may cause different stability towards thermal
stress and consequently distortions in the results of
relative quantification (Moreano et al., 2005a). Milling
fractions produced from genetically modified Bt176 corn
were thermally treated and analyzed by applying two
established quantitative assays showing differences
between the lengths of the recombinant and reference
target sequences (method A: DlAZK25 bp, method B:
DlBZC16 bp; values related to the amplicon length of
the reference gene). Data obtained by the application of
method A resulted in underestimated recoveries of GMO
contents in the samples of heat-treated products,
reflecting the favored degradation of the longer target
sequence used for the detection of the transgene. In
contrast, the application of method B resulted in an
Fig. 3. Relative quantification of DNA from corn Bt-176 in samples
from intermediate stages of the process of ethanol production; NQ,
not quantifiable. (Moreano, 2005; Moreano et al., 2005b).
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
increasing overestimation of GMO contents (Fig. 2). The
degree of distortion of the relative quantification results
was lower than that observed in the previous method,
due to the smaller absolute length difference between
the targeted sequences for the recombinant DNA and
the reference gene.
In a similar approach, a mixture of maize grits with a
defined proportion of GMO-derived material (10%) was
used in a lab-scaled process for the production of ethanol.
This example was chosen because it comprises a series of
processes potentially contributing to DNA degradation
(mechanical stress, enzymatic hydrolyses, microbial
fermentation, thermal treatment). The starting material as
well samples from the stages of mashing, enzymatic
liquefaction and saccharification, mash fermentation and
mash distillation were analyzed using three different realtime PCR systems (Fig. 3). Systems exhibiting differences
in lengths between the target and reference sequence
resulted in underestimation (Dl!0) or overestimation
(DlO0) of the adjusted GMO content. Only after
minimizing the difference to DlZK2 bp, a nearly constant
recovery of the actual proportion of GMO-derived material
was achieved even at high levels of processing (Moreano,
2005; Moreano, Busch, & Engel, 2005b).
Multiplex assays: ligation-dependent probe
amplification (LPA)
In order to meet the challenge of the steadily
increasing number of GMO, various multiplex assays
have been developed, allowing the simultaneous detection of several GMO in a single PCR reaction (Forte et
al., 2005; Germini, Zanetti, Salati, Rossi, Forré, Schmid,
493
et al., 2004; Hernandez et al., 2005; James, Schmidt,
Wall, Green, & Masri, 2003; Matsuoka et al., 2001;
Onishi et al., 2005). Additionally, qualitative applications for identification of GMO in food have been
introduced using detection via microarray technology
(Germini, Mezzelani, Lesignoli, Corradini, Marchelli,
Bordoni et al., 2004, 2005; Xu et al., 2005). A
combined assay of multiplex polymerase chain reaction
and ligation detection reaction coupled with microarray
detection was developed by Bordoni, Germini, Mezzelani, Marchelli, and De Bellis (2005) and applied by
Peano et al. (2005) to the traceability of GMO in foods.
A novel multiplex quantitative DNA array-based PCR
has been presented by Rudi, Rud, and Holck (2003) for
the quantification of transgenic maize in food and feed.
One of the latest developments in multiplex
approaches for quantitative analysis is the technique of
ligation-dependent probe amplification (LPA). Originally,
ligation-dependent PCR has been applied to the relative
quantification of DNA in the field of medical diagnostics
(Schouten et al., 2002). The suitability of this method for
the detection and relative quantification of GMO in food
samples has been demonstrated using commercially
available maize standards (Moreano, Ehlert, Busch, &
Engel, 2006). A synthetic probe set is used for the
detection of target sequences to avoid complex cloning
and preparation steps required for the isolation of single
stranded DNA probes. These sequence-specific probes
contain a target-specific hybridization site and identical
primer binding sites (PBS) at their 5 0 - and 3 0 -ends,
respectively. In case of successful hybridization to
adjacent sites of the target sequence, the probes are
ligated by a thermostable ligase. The use of spacer
Fig. 4. Electropherogram obtained by LPA from a sample containing Roundup Ready soya, maize MON 810 and hybrid molecules (Moreano,
Pecoraro, Bunge, & Busch 2005). Simultaneous detection of reference genes in the genome from maize (HMGa-gene), soya (Le1-gene) and
rapeseed (ACCg8-gene), the CaMV 35S-promotor and the junction 35S-pat gene as well as event-specific regions of the transgenic maize line
MON 810 and Roundup Ready soya. Non-assigned signals correspond to the used length standards.
494
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
sequences between hybridization sites and PBS assures
ligation products with lengths characteristic for each of
the target DNA. In the second step of the reaction, the
ligation products are amplified competitively using one
labelled pair of primers. Labelled PCR amplicons are
finally separated by capillary electrophoresis and detected
via laser-induced fluorescence.
The use of one pair of universal primers in the LPA
method avoids one of the major difficulties of multiplex
PCR applications, being the complexity of amplification
reaction because of the use of multiple pairs of primers.
In contrast to multiplex PCR reactions, whose subsequent
extensibility is limited, the application of this novel
approach offers great flexibility due to its modular
system that can be complemented with further probes
to broaden the range of target sequences. The use of
synthetic oligonucleotides as probes and the employment
of classical thermocycler and detection methods enable
the implementation of the technique in commonly
equipped laboratories.
Detection using capillary gel electrophoresis via laserinduced fluorescence (CGE-LIF) represents a very sensitive
and rapid separation and detection approach in automated
manner. Different amplicons can be distinguished by size
using either labelled PCR primers or DNA intercalating
dyes. The better sensitivity and resolution of CGE-LIF
compared to agarose gel electrophoresis has been demonstrated (Garcia-Canas, Gonzalez, & Cifuentes, 2004).
Capillary electrophoresis is a useful tool for optimization
of multiplex PCR reactions by the possibility to obtain
quantitative data in form of peak areas/heights (Butler,
Riutberg, & Vallone, 2001).
Currently, the LPA-approach allows the screening of
seven different targets in a one-tube assay (Fig. 4).
Synthetic DNA standards
The steadily increasing number of transgenic crop
lines worldwide demands the implementation of appropriate systems for the detection and quantification of a
broad spectrum of GMO. This requires the availability of
respective certified reference standard materials. Taking
into consideration that the vast majority of transgenic
crops have not been submitted (and are not foreseen) for
authorization in the EU, the investigation of the
occurrence of material derived from non-authorized
GMO in the food and feed chain represents a principal
task for surveillance authorities. One of the most
challenging problems is given by the hesitant development of reference standards for qualitative and/or
quantitative analyses. Commercially available reference
standards do not cover the entire spectrum of authorized
GMO and there is (per se) a complete lack of reference
material for the analysis of non-authorized transgenic
crops.
Novel analytical strategies are based on the design of
synthetic DNA-standards in combination with the set-up
of respective real-time PCR systems for the quantitative
screening of construct-specific sequences (Moreano,
Fig. 5. Synthesis of hybrid DNA-molecules to be used as reference standards for the quantitative screening of GMO. (A) The synthesis is initiated
by the amplification of the targeted recombinant and taxon-specific sequences in separate reactions. Amplifications are carried out under
application of bipartite primers possessing complementary overhanging sequences (OVH and OVH 0 ). (B) Purified amplicons are mixed and
used as template in a second PCR, where the self-priming activity of the overhang sequences initiate the generation of hybrid molecules
(Pardigol et al., 2003; Moreano et al., 2005c).
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
495
Fig. 6. Amplification plots of dilution series of synthetic DNA-standards (105–100 molecules/mL) in duplex real-time PCR assays. Calibration
curves, described by the given equations, were gained by plotting the Ct-values determined for the GMO-specific and rapeseed-specific
targets against the logarithm of the copy numbers in the respective standard dilution and used for the quantification of unknown samples
(Moreano et al., 2005c).
Pecoraro, Bunge, & Busch, 2005). The approach was
applied exemplarily to the analysis of genetically
modified rapeseeds, where both the number of transformation events as well as the lack of reference standards
represents significant drawbacks for surveillance
laboratories.
Two real-time PCR assays were designed for the
purpose of quantitative screening. The assays allow the
simultaneous detection of construct-specific junction
regions in transgenic rapeseed lines (P-35S/pat—gene
junction in LibertyLinke lines; bar—gene/T-g7 junction
in SeedLinke lines) as well as the detection of a
rapeseed-specific acetyl-CoA carboxylase reference gene
in a duplex reaction. The detection of taxon-specific
reference genes is essential for the normalization of
quantification results.
As illustrated in Fig. 5, the synthesis of DNA-standards
for the relative quantification of recombinant sequences can
be achieved according to a novel PCR technique allowing
the amplification of hybrid molecules (Pardigol, Guillet, &
Pöpping, 2003). The synthetic standards carry GMOspecific and taxon-specific target sequences at a 1:1 ratio
and can be employed for the generation of dilution series
with defined copy number concentrations.
The applicability of dilution series of the hybrid
molecules as quantification standards in real-time PCR
was demonstrated by their performance in a concentration range between 105 and 100 molecules/mL.
Reaction conditions of the duplex assays were optimized
in order to allow an accurate quantification of
recombinant DNA related to the copy number of the
taxon-specific reference gene at GMO-contents of 0.9%.
This was achieved by limiting the concentration of
taxon-specific primers, while considering that the
performance of the DNA-standards in the duplex
reactions (i.e. Ct-values and slopes of the standard
curves) may not differ from the performance delivered
by simplex assays. As shown exemplarily in Fig. 6,
primer limitation resulted in reduced final rapeseedspecific signal intensities but did not lead to a shift of
the determined Ct-values.
This analytical strategy represents a valuable tool for
purposes of surveillance testing of GMO in food and feed
products. The application of construct-specific real-time
PCR assays allow the detection of a broad spectrum of
GMO at a moderate level of specificity in order to avoid
false positive results, such as expected in the case of the
detection of regulatory sequences or gene coding regions
alone.
Outlook
Data gained from the performance assessment of the
assays described deliver important criteria for future
method development and for the design of validation
studies. Especially in regard to the establishment of
standardized international protocols for the quantitative
analysis of GMO in foods, validation studies cannot be
restricted to the assessment of trueness and precision in
(unprocessed) certified reference materials, since such
performance parameters may not be unconditionally
transferable to results obtained from the analysis of
496
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
composed and/or processed samples. Therefore,
procedures for the validation of quantitative assays for
the surveillance testing of products within the food chain
must include experiments demonstrating that neither food
composition nor processing will result in an alteration of
relative quantification results. Future standard protocols
for the determination of GMO contents in processed
products should target recombinant and taxon-specific
sequences of nearly equal lengths. Otherwise, the scope
of the analytical approaches should be clearly limited to
the determination of GMO contents in unprocessed
samples.
In combination with synthetic quantification standards,
quantitative screening assays can be used to gain
important information about the compositions of samples.
The application of such assays will contribute to avoid
the unnecessary application of event-specific quantification methods, e.g. given the case that results of
quantitative screening predict only traces of recombinant
DNA to be present in a sample, or if the delivered copy
numbers of the reference gene are not sufficient to allow
a statistically secured quantification of the recombinant
DNA.
References
Alexander, R. J. (1987). Corn dry milling: Processes, products, and
applications. In S. A. Watson, & P. E. Ramstad (Eds.), Corn:
Chemistry and technology. Saint Paul, MN: American Association of Cereal Chemists.
Anklam, E., Gadani, F., Heinze, P., Pijnenburg, H., & Van den Eede,
G. (2002). Analytical methods for detection and determination
of genetically modified organisms in agricultural crops and
plant-derived food products. European Food Research and
Technology, 214, 3–26.
Bauer, T., Weller, P., Hammes, W. P., & Hertel, C. (2003). The effect
of processing parameters on DNA degradation in food. European
Food Research and Technology, 217, 338–343.
Berdal, K. G., & Holst-Jensen, A. (2001). Roundup ready soybean
event-specific real time quantitative PCR assay and estimation of
the practical detection and quantification limits in GMO
analyses. European Food Research and Technology, 213, 432–
438.
Bordoni, R., Germini, A., Mezzelani, A., Marchelli, R., & De
Bellis, G. (2005). A microarray platform for parallel detection
of five transgenic events in foods: A combined polymerase
chain reaction–ligation detection reaction–universal array
method. Journal of Agricultural and Food Chemistry, 53, 912–
918.
Broll, H., Zagon, A., Butschke., & Grohmann L. GVO Analytik:
Validierung und Ringversuche. GMO Analytik heute. Symposium organized by Scil Diagnostics GmbH and GeneScan
Europe AG. Frankfurt am Main, Germany, 23rd January,
2002.
Butler, J. M., Riutberg, C. M., & Vallone, P. M. (2001). Capillary
electrophoresis as a tool for optimization of multiplex PCR
reactions. Fresenius Journal of Analytical Chemistry, 369(3–4),
200–205.
EC (2003a). Regulation (EC) No. 1829/2003 of the European
Parliament and of the Council of 22nd September 2003 on
genetically modified food and feed. Official Journal of the
European Union, L 268/1.
EC (2003b). Regulation (EC) No 1830/2003 of the European
Parliament and of the Council of 22nd September 2003
concerning the traceability and labelling of genetically modified
organisms and the traceability of food and feed products
produced from genetically modified organisms and amending
Directive 2001/18/EC. Official Journal of the European Union, L
268/24.
Forte, V. T., Di Pinto, A., Martino, C., Tantillo, G. M., Grasso, G., &
Schena, F. P. (2005). A general multiplex-PCR assay for the
general detection of genetically modified soya and maize. Food
Control, 16, 535–539.
Garcia-Canas, V., Gonzalez, R., & Cifuentes, A. (2004). Sensitive
and simultaneous analysis of five transgenic maizes using
multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence. Electrophoresis,
25(14), 2219–2226.
Germini, A., Mezzelani, A., Lesignoli, F., Corradini, R., Marchelli,
R., Bordoni, R., et al. (2004). Detection of genetically modified
soybean using peptide nucleic acids (PNAs) and microarray
technology. Journal of Agricultural and Food Chemistry, 52(14),
4535–4540.
Germini, A., Rossi, S., Zanetti, A., Corradini, R., Fogher, C., &
Marchelli, R. (2005). Development of a peptide nucleic acid
array platform for the detection of genetically modified
organisms in food. Journal of Agricultural and Food Chemistry,
53(10), 3958–3962.
Germini, A., Zanetti, A., Salati, C., Rossi, S., Forré, C., Schmid, S.,
et al. (2004). Development of a seven-target multiplex PCR for
the simultaneous detection of transgenic soybean and maize in
feeds and foods. Journal of Agricultural and Food Chemistry,
52(11), 3275–3280.
Hernandez, M., Rodriguez-Lazaro, D., Zhang, D., Esteve, T., Pla,
M., & Prat, S. (2005). Interlaboratory transfer of a PCR multiplex
method for the simultaneous detection of four genetically
modified maize lines: Bt11, MON810, T25, GA21. Journal of
Agricultural and Food Chemistry, 53(9), 3333–3337.
Höhne, M., Santisi, C. R., & Meyer, R. (2002). Real-time
multiplex PCR: An accurate method for the detection and
quantification of 35S-CaMV promoter in genetically modified
maize-containing food. European Food Research and Technology, 215, 59–64.
Holck, A., Va, M., Didierjean, L., & Rudi, K. (2002). 5 0 -Nuclease
PCR for quantitative event-specific detection of the genetically
modified Mon810 MaisGuard maize. European Food Research
and Technology, 214, 449–453.
Hübner, P., Studer, E., & Lüthy, J. (1999). Detection of genetically
modified organisms in food. Nature Biotechnology, 17, 1137–
1138.
Hupfer, C., Hotzel, H., Sachse, K., & Engel, K.-H. (1998). Detection
of the genetic modification in heat-treated products of Bt maize
by polymerase chain reaction. Zeitschrift für LebensmittelUntersuchung und -Forschung A, 206, 203–207.
Hupfer, C., Hotzel, H., Sachse, K., Moreano, F., & Engel, K.-H.
(2000). PCR-based quantification of genetically modified Bt
maize: single-competitive versus dual-competitive approach.
European Food Research and Technology, 212(1), 95–99.
Hupfer, C., Mayer, J., Hotzel, H., Sachse, K., & Engel, K.-H. (1999).
Effect of ensiling on PCR-based detection of genetically modified
Bt maize. European Food Research and Technology, 209(5),
301–304.
Hupfer, C., Schmitz-Winnenthal, J., & Engel, K.-H. (1999).
Detection of genetic change in transgenic potatoes during the
distillery process in ethanol production. Lebensmittelchemie,
53(1), 13–14.
K.-H. Engel et al. / Trends in Food Science & Technology 17 (2006) 490–497
ILSI Europe (2001). Novel Food Task Force in collaboration with the
European Commission’s Joint Research Center (JRC) and ILSI
International Food Biotechnology Committee. Method
development in relation to regulatory requirements for detection
of GMOs in the food chain. ILSI Europe report series. ILSI Europe,
Brussels, 2001.
ISO/DIS. (2003). Foodstuffs—methods of analysis for the detection
of genetically modified organisms and derived products—
quantitative nucleic acid based methods. European Committee
for Standardization (21570: 2003).
James, D., Schmidt, A.-M., Wall, E., Green, M., & Masri, S. (2003).
Reliable detection and identification of genetically modified
maize, soybean, and canola by multiplex PCR analysis. Journal
of Agricultural and Food Chemistry, 51, 5829–5834.
Jonas, D. A., Elmadfa, I., Engel, K.-H., Heller, K. J., Kozianowski, G.,
König, A., et al. (2001). Safety considerations of DNA in food.
Annals of Nutrition and Metabolism, 45(6), 235–254.
Klein, J., Altenbuchner, J., & Mattes, R. (1998). Nucleic acid and
protein elimination during the sugar beet manufacturing process
of conventional and transgenic sugar beets. Journal of Biotechnology, 60, 145–153.
Matsuoka, T., Kuribara, H., Akiyama, H., Miura, H., Goda, Y.,
Kusakabe, Y., et al. (2001). A multiplex PCR method of
detecting recombinant DNAs from five lines of genetically
modified maize. Journal of Hygiene Society Japan, 42(1),
24–32.
Miraglia, M., Berdal, K. G., Brera, C., Corbisier, P., Holst-Jensen, A.,
Kok, E. J., et al. (2004). Detection and traceability of genetically
modified organisms in the food production chain. Food and
Chemical Toxicology, 42, 1157–1189.
Moreano, F. (2005). Development of techniques for the quantification of DNA from genetically modified organisms in processed
foods. Dissertation Technical University Munich, Germany.
Moreano, F., Busch, U., & Engel, K.-H. (2005a). Distortion of
genetically modified organism quantification in processed foods:
Influence of particel size compositions and heat-induced DNA
degradation. Journal of Agricultural and Food Chemistry, 53,
9971–9979.
Moreano, F., Busch, U., & Engel, K.-H. (2005b). Einfluss der
Lebensmittelverarbeitung auf die GMO-Analytik. In H.-U.
Waiblinger, & U. Busch (Eds.), Neue Regelungen für gentechnisch veräenderte Lebensmittel und Futtermittel. Schriftenreihe
Lebensmittelchemische Gesellschaft-Band 27. Hamburg,
Germany: Behr’s Verlag.
Moreano, F., Ehlert, A., Busch, U., & Engel, K. H. (2006). Ligationdependent probe amplification for the simultaneous eventspecific detection and relative quantification of DNA from two
genetically modified organisms. European Food Research and
Technology, 222, 479–485.
Moreano, F., Pecoraro, S., Bunge, M., & Busch, U. (2005).
Development of synthetic DNA-standards for the quantitative screening of different genetically modified rapeseed
lines via real-time PCR. In Proceedings of the Euro Food
Chem XIII Conference, macromolecules and their
degradation products in food—physiological, analytical and
technological aspects, Hamburg (pp. 154–158), Vol. 1, 21–
23 September 2005.
Moser, M. A., Kniel, B., Schmitz-Winnenthal, J., Hupfer, C., & Engel,
K.-H. (1999). Einfluß verfahrenstechnische Parameter auf den
analytischen Nachweis gentechnisch veräenderter Zutaten in
Backwaren. Getreide Mehl und Brot, 6, 1–8.
497
Onishi, M., Matsuoka, T., Kodama, T., Kashiwaba, K., Futo, S.,
Akiyama, H., et al. (2005). Development of a multiplex
polymerase chain reaction method for simultaneous detection of
eight events of genetically modified maize. Journal of Agricultural and Food Chemistry, 53(25), 9713–9721.
Pardigol, A., Guillet, S., & Pöpping, B. (2003). A simple procedure
for quantification of genetically modified organisms using hybrid
amplicon standards. European Food Research and Technology,
216, 412–420.
Peano, C., Bordoni, R., Gulli, M., Mezzelani, A., Samson, M. C., De
Bellis, G., et al. (2005). Multiplex polymerase chain reaction and
ligation detection reaction/universal array technology for the
traceability of genetically modified organisms in foods.
Analytical Biochemistry, 346(1), 90–100.
Pietsch, K., & Waiblinger, H.-U. (2001). Quantification of
genetically modified soybeans in food with the LightCycler
system. Rapid cycle real-time PCR (pp. 385–389).
Rooney, L. W., & Serena-Saldivar, S. O. (1987). Food uses of whole
corn and dry-milled fractions. In S. A. Watson, & P. E. Ramstad
(Eds.), Corn: Chemistry and technology. Saint Paul, MN:
American Association of Cereal Chemists, Inc.
Rudi, K., Rud, I., & Holck, A. (2003). A novel multiplex quantitative
DNA array based PCR (MQDA-PCR) for quantification of
transgenic maize in food and feed. Nucleic Acids Research,
31(11), e62.
Schouten, J. P., McElgunn, C. J., Waaijer, R., Zwijnenburg, D.,
Diepvens, F., & Pals, G. (2002). Relative quantification of 40
nucleic acid sequences by multiplex ligation-dependent probe
amplification. Nucleic Acids Research, 30(12), e57.
Spiegelhalter, F., Lauter, F. R., & Russell, J. M. (2001). Detection of
genetically modified food products in a commercial laboratory.
Journal of Food Science, 66(5), 634–640.
Straub, J. A., Hertel, C., & Hammes, W. P. (1999). Limits of a PCRbased detection method for genetically modified soya beans in
wheat bread production. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 208, 77–82.
Taverniers, I., Windels, P., Van Bockstaele, E., & De Loose, M.
(2001). Use of cloned DNA fragments for event-specific
quantification of genetically modified organisms in pure and
mixed food products. European Food Research and Technology,
213, 417–424.
Terry, C. F., & Harris, N. (2001). Event-specific detection of Roundup
Ready soya using two different real time PCR detection
chemistries. European Food Research and Technology, 213,
425–431.
Vaitilingom, M., Pijnenburg, H., Gendre, F., & Brignon, P. (1999)
. Real-time PCR detection of genetically modified Maximizer
maize and Roundup Ready soybean in some representative
foods. Journal of Agricultural and Food Chemistry, 47, 5261–
5266.
Wiseman, G. (2002). State of the art and limitations of quantitative
polymerase chain reaction. Journal of AOAC International, 85(3)
, 792–796.
Wurz, A., Bluth, A., Zelz, P., Pfeifer, C., & Willmund, R. (1999).
Quantitative analysis of genetically modified organisms (GMO)
in processed food by PCR based methods. Food Control, 10,
385–389.
Xu, X., Li, Y., Zhao, H., Wen, S.-Y., Wang, S.-Q., Huang, J., et al.
(2005). Rapid and reliable detection and identification of GM
events using multiplex PCR coupled with oligonucleotide
microarray. Journal of Agricultural and Food Chemistry, 53(10),
3789–3794.