Validated Method for Quantification of Gentically Modified
Organisms in Samples of Maize Flour
RENATE KUNERT,*,†JOHANNES S. GACH,†KAROLA VORAUER-UHL,†
EDWIN ENGEL,§AND HERMANN KATINGER†
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences,
Muthgasse 18, A-1190 Vienna, Austria, and Agrana Maissta ¨rkefabrik, Raiffeisenweg 2-6,
A-4082 Aschach, Austria
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material
is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or
raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR)
amplification has easier sample preparation and detection limits are lower. Of the different methods
of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient
DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green
detection method showed that the method is highly sensitive to sample matrices and genomic DNA
content influencing the interpretation of results. Therefore, this paper describes a real-time DNA
quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with
detection limits of lower than 18 copies per sample applicable and comparable to highly purified
plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated
with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard
KEYWORDS: 35S promoter; GMO; maize; TaqMan; ValiData
Maize (Zea mays L.) is grown primarily for its kernel, which
is largely refined into products used in a wide range of food,
medical, and industrial goods. Only a small amount of whole
maize kernel is consumed by humans. Maize oil is extracted
from the germ of the maize kernel, and maize is also used in
the manufacture of starch. Refined maize products, sweeteners,
starch, and oil are abundant in processed foods such as breakfast
cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as
animal feed, with roughly 80% of the crop fed to livestock.
Animals that feed on maize include cattle, pigs, poultry, sheep,
goats, fish, and companion animals.
The European Union (EU) dictates the scientific evaluation
for the permission of genetically modified organisms (GMO)
in human food as well as feeding stuff to generate a concise
and transparent system. Since 2004 all food comprising GMO
or prepared from GMO has to be indicated independently of
the traceability of the GMO in the final product. This strategy
is encoded in EU Regulation 1830/2003 regulating the labeling
and retracing of the whole production process. In the case of
genetically modified material that is not approved in the EU,
despite a positive scientific estimation, the threshold value to
be inserted into food is 0.5%. This relative percentile quantifica-
tion requires the absolute quantification of the distinct DNA
species in the sample and is usually accomplished by spectro-
photometrical DNA detection also used in our laboratory. An
alternative is introduced by Hernandez et al. (1), who reported
different endogenous genes of the species as reference point
for DNA quantification in an unknown variety (2).
The principal traits in most of the GM maize breeds are
herbicide and insect tolerance (3) under control of Cauliflower
mosaic virus 35S promoter (CaMV 35S) (4). In this paper we
describe a method for the quantification of the 35S promoter
in maize flour, based on a plasmid standard curve with a
sensitivity of 18 copies per sample. The advantage of this
validated method is the independence from the sample matrix
due to TaqMan probes and the easy handling of standards. In
our example we used a 162 bp amplicon of the 35S promoter
for quantification and confirmed the sequence homology for
different GM maize species to guarantee equal PCR efficiencies
necessary for correct quantification. The real-time (RT) PCR
amplification is described by an exponential function that is
reduced to a first-order function by taking the logarithm of the
starting concentration. Therefore, the results have to be validated
carefully; otherwise, the variation of final results is too high.
The interpretation of the raw data and conversion to the gene
copy number of starting material are discussed in detail.
MATERIALS AND METHODS
BCR Standards. Cornmeal samples of maize BT11 (5% GMO),
NK603 (5% GMO), and MON810 (5% GMO) reference standards were
received from the Bureau Communautaire de Re `fere `nce (BCR;
developed at the Institute for Reference Materials and Measurements,
Geel, Belgium, commercialized by Fluka), purified by the CTAB
method (5) and diluted to 100 ng/µL.
Genomic DNA sample, AM32, was provided by Agrana Mais-
sta ¨rkefabrik (Aschach, Austria) in a concentration of 100 ng/µL. In
brief, 2 g of homogenized cornmeal was diluted in 15 mL of CTAB
extraction buffer (20 g/L hexadecyltrimethylammonium bromide, 81.8
g/L NaCl, 5.8 g/L EDTA, pH 8.0), incubated for 60 min at 65 °C, and
centrifuged for 10 min at 12000g. Five milliliters of the upper layer
was extracted with 4 mL of chloroform. After 30 s of mixing, the
mixture was centrifuged for 15 min at 12000g. Four milliliters of
supernatant was transferred into a new tube, and 2 volumes of CTAB
precipitation solution (5 g/L CTAB, 2.34 g/L NaCl) was added. The
mixture was incubated for 60 min at room temperature and then
centrifuged for 10 min at 12000g. The supernatant was removed, and
the precipitate was dissolved in 1.2 M NaCl and chloroform extracted;
2.5 mL of the upper layer (aqueous phase) was transferred to a new
tube, and 0.7 volume of 2-propanol was added. After centrifugation,
the resulting DNA pellet was washed with ice-cold 70% ethanol and
centrifuged. The ethanol was discarded, and residual ethanol was
removed in a vacuum exsiccator; the DNA pellet was resuspended in
100-500 µL of TE buffer.
Plasmid standards were generated by amplification of 35S promoter
with 35S-7245 sense 5′-gaattcccgacagtggtcccaaagatgg-3′ and 35S-7406
antisense 5′-gcggccgcatatagaggaagggtcttgc-3′ primers and cloned into
the EcoRI/NotI opened pBluescript (Stratagene, La Jolla, CA) vector.
Inserts of standard genomic DNAs (NK603, BT11, and MON810) were
sequenced using the standard chain termination method (6) and aligned
to the 35S promoter of cauliflower mosaic virus genome (Genbank
accession no. V00140, J02046). Plasmids containing Escherichia coli
Top 10 were propagated in ampicillin containing LB medium, and
plasmids were purified via Qiagen Tip 100 (Qiagen, Norway).
Establishment of Plasmid Standard Stock Solution. Plasmid DNA
was quantified spectrophotometrically at 260 nm, diluted to a concen-
tration of 10E4 copies/µL (33.9 × 10-6ng/µL), and stored in aliquots
at -20 °C.
RT-PCR with TaqMan Probes. RT-PCR assays were carried out
in an iCycler (Bio-Rad Laboratories, Hercules, CA), using the TaqMan
system in a final volume of 25 µL; all primers and probes were
synthesized at Invitrogen. The reaction mix included 12.5 µL of
iQsupermix (Bio-Rad) 0.5 µM forward primer, 35S-7248 sense
5′-ACAGTGGTCCCAAAGATGGA-3′, 0.5 µM antisense primer, 35S-
7396 antisense, 5′-AGGGTCTTGCGAAGGATAGT-3′, and 0.5 µM
fluorogenic probe, 35S-7269 sense-probe, 5′-CCCCACCCACGAG-
GAGCATCG-3′, labeled with 6-carboxyfluorescein (FAM) at the 5′-
end and with the fluorescent quencher dye 6-carboxytetramethyl-
rhodamin (TAMRA) at the 3′-end. Primers used in this assay were
designed using Primer 3 software (7). Conditions for amplification were
10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Each
sample including all controls and points from the standard curve was
quantified in triplicates.
Validation of Results. Statistical control of the linear model was
performed according to International Conference on Harmonisation
(ICH) guidelines (ICH Harmonised tripartite Guidelines, 1996 ICH
steering committee, “Validation of analytical procedures: methodology
Q2B”) with the ValiData software (8).
Different parameters have been calculated to validate this method;
those specifying the standard curve and the sample detection are
1. The linear range of the calibration standard curve equation was
tested according to ValiData.
2. The limit of detection (LOD) as well as the limit of quantification
(LOQ) was estimated by ValiData according to the calibration curve
method as a multiple of the standard deviation.
3. The variance was detected for highest and lowest concentration
of the curve and judged at 95 and 99% significance levels in ValiData.
4. The relative standard deviation (% CV) and the recovery (% r)
are defined as
where m is the mean and σ is the standard deviation.
Sequence Homology of GMO Standards. Plasmids contain-
ing the amplified 162 bp fragment of the 35S promoter were
sequenced, and 100% homology was shown for all inserts
amplified from NK603, BT11, and MON810 according to the
cauliflower mosaic virus genome (9, 10). Additionally, positively
tested DNA samples provided by Agrana Maissta ¨rkefabrik were
also shown to have identical 35S promoter fragments.
Verification of Standards. The plasmid standard calibration
curve was verified by comparison of diluted plasmids with 5%
GMO MON810 genomic DNA samples. Plasmid standards
containing 2000, 200, and 20 copies and MON810 5% GMO
genomic DNA samples (1800, 180, and 18 copies) were used
as template in the same RT-PCR experiment, and the plasmid
standards as well as MON810 standards comprise the standard
curve. Figure 1 shows the correlation coefficient and the slope
of the curve as well as the interception when using all six
different standard sample concentrations analyzed in triplicate.
The correlation coefficient was estimated to be 0.98, giving clear
evidence for the accuracy of the independently prepared samples
and the method. The amplification efficiency is demonstrated
by the slope of the curve, indicating 87% efficiency resulting
from a slope of -3.7. Validation of these results with ValiData
confirmed the linearity of the calibration curve. Additionally,
the same conclusion gave the check of variances of highest and
lowest values at 95 and 99% levels of significance. The LOD
and the LOQ were indicated by 2.3 and 3.9 copies, respectively.
The residual analysis of all calibration points is shown in
Figure 2, showing no deviation of the Gaussian distribution
and no trend across the calibration curve. Figure 3 describes
the calibration interval and the prediction interval based on the
data of the standard curve.
Quantification of Sample AM32. The gene copy number
of 35S promoter in sample AM32 was determined in three
different experiments. Table 1 summarizes the results obtained
using the plasmid calibration curve. The average result of these
analyses gave 25 copies of 35S promoter in 200 ng of genomic
DNA, resulting in 0.07% GM maize in sample AM32.
Figure 1. Amplification plot of real-time calibration curve including plasmid
standards and GMO standards. The correlation between cycle number
(ct) and initial copy number of 35S promoter is described by the interception
of 40.4, the correlation coefficient (R2) 0.98), and slope of the curve:
−3.77. Dots indicating plasmid standards are described by log starting
concentrations of 3.3, 2.3, and 1.3; those of the 5% GMO MON 810
have log starting quantities of 3.26, 2.26, and 1.26.
% CV )100σ
% r )
predicted - copies
Assessment of Matrix. In this experiment plasmid standards
with a concentration of 2000, 200, or 20 copies per 2 µL of
template were used for quantification. Two artificial samples
were generated by mixing plasmid standards and genomic corn
DNA samples. Artificial sample 1 (AS1) was generated by
mixing 18 µL of AM32 (with a content of <0.5% GMO) with
2 µL of the 1000 copies/µL plasmid standard; AS2 consisted
of 5 µL of BT11 DNA (5% GMO BT11 in corn meal mixture)
plus 5 µL of 100 copies/µL plasmid standard; 2 µL of DNA
template was used in each experiment for artificial samples,
pure GMO samples, and plasmid standards. In theory, 2 µL of
the BT11 sample contains 360 copies of the 35S promoter
amplicon in the dilution 1:5.
Table 2 explains the samples, the raw data indicated by
threshold cycles, and predicted copy number of 5% GMO BT11
standard and artificial samples as well as the results generated
by the plasmid calibration curve analyzed in triplicates (BT11,
AS1, and AS2) and mean values thereof. The concentration of
AS1 is not defined because we do not know the distinct
concentration of this real sample. We have determined the
portion of GMO in sample AM32 with 25 copies per 200 ng of
DNA, clearly indicated to be lower than the 0.5% GMO limit.
Calculation with this analyzed value gives a prediction of 222.5
copies for AS1. In the case of the sample 5% GMO BT11 the
theoretical value is 360 copies in this preparation, and the
generated results were dispersed between 249 and 430 copies.
However, the mean value indicated good accordance with the
predicted value. Similar dispersion was evident for the samples
AS1 ranging from 149 to 285 copies in this approach and AS2
with a dispersion of 799-1300. Despite this dispersion, mean
values of independent analysis of AS1, AS2, and 5% GMO
BT11 gave good accordance with the predicted gene copy
numbers. All data generated in this experiment were validated
for their statistical relevance.
Validation of Data. The RT-PCR interpretation of initial gene
copy number in genomic DNA samples occurs via a linear
standard curve generated by logarithms of starting concentration
of the target gene correlated to the threshold cycle, indicated
by the detection of a distinct fluorogenic signal. This standard
curve was verified by comparison of first- to second-order
curves, resulting in confirmation of the linear range of the
The LOD and LOQ were defined and allow a quantification
of GMO lower than 0.5% GMO in 200 ng of genomic DNA
indicated by a threshold of 182 copies. However, the approved
LOQ allows the detection of <0.1% GMO in 200 ng of maize
The matrix exploration experiment was validated for accuracy
and recovery, yielding 87.6-100.9% recovery of predicted
values, indicating a very efficient quantification of samples
The statistical spread is demonstrated best by the relative
standard deviation (RSD) and is calculated to be <1.8% for ct
values in three independent measurements with no indication
for abnormal statistical variation (Table 2). The read-out of these
ct values is the “log starting quantity” (compare Figure 1), and
the RSD was calculated to be 3.61-7.21% in our quantification
model, a rather good result for such a complex method.
However, the PCR is an exponential method, and inversion of
the logarithm of starting quantity leads to a higher spread of
the final results also shown in Table 2. In case of trace amounts
of GMO in maize flour it is not recommended to state the
absolute gene copy number, but rather to define the threshold
with <0.5% GMO in the sample (compare Table 1).
Taken together this validated quantification meets all demands
for the absolute quantification of GMO in maize flour with
amounts >0.5% of the transgene.
Figure 2. Analysis of residues of the calibration curve.
Figure 3. Calibration curve with confidential interval and interval of
Table 1. Quantification of 35S Promoter Gene Copy Numbers in 200
ng of AM32 Genomic DNA in Three Independent Experiments in
Triplicate and Quadruplicate, Respectively
single values (copies)
Table 2. Statistical Examination of the RT-PCR Quantification in
5% GMO BT11 1:5AS1AS2
Statistical Analysis of ct Values
30.93 31.77 31.05 32.46 31.56 32.64 29.22
Statistical Analysis of the Log Gene
Copy Number Reflecting the Raw Data
predicted log copies
Statistical Analysis of Final Copy Number
159 1411300 799
aSamples used were 5% GMO BT11; AS1, consisting of 5% GMO BT11 and
plasmid standard; and AS2, consisting of sample AM32 and plasmid standard.
Statistical parameters [mean value, relative standard deviation (RSD), and recovery]
were calculated for the results demonstrated as ct values, the logarithm of gene
copy number, and copies per sample.bThreshold cycles.cSingle values from
analyses in triplicate.
In this paper we describe a method for the detection of GMO
in real maize samples according to EU Regulation 1830/2003
based on a 0.5% limit. Different strategies to detect transgenic
DNA have been described (e.g., amplification of fragments of
the 35S promoter, the NOS ending, and the junction of coding
sequence and regulatory sequence). However, any of these
methods is uncertain, and the quantification depends on the
determination of transgene and the total copy number of DNA
The relevant issues in real-time quantification of the GMOs
are the choice of adequate primers, probes, and evaluation
model, the appropriate standard material, and the DNA isolation
method. The DNA extraction method should be cost- and time-
effective, especially in the case of high amounts of samples.
Previous examinations of different commercially available kits
based on silica gel, magnetic beads, and precipitation were
compared to lysis and precipitation with CTAB, showing that
the conventional CTAB purification gave highest yield with
sufficient PCR amplification protocols (12). The standard curves
are predominantly generated from BCR GMOs in concentrations
between 5 and 0.1% GMO in the corn meal (13, 14). The use
of genomic DNA for the calibration curve is essential for the
SYBR green method, where the fluorophor intercalates with
the entire double-stranded DNA generated during amplification
and unspecific amplicons in trace amounts may falsify the
results. Therefore, the SYBR green method is sensitive to matrix
effects, and it is not possible to dilute one distinct concentration
of the BCR standard for the generation of a standard curve (data
not shown), but rather a whole series of standards has to be
prepared, controlled for purity, and quantified. Besides the real-
time experiments we have also sequenced the 162 bp long
amplicon of CaMV 35S promoter and found 100% homology
for BT11, NK603, and MON810, giving evidence for the
quantification of real samples with these primers. Additionally,
we compared the amplicons of the invertase exon from three
of our breedings with the Genbank entry (GI 1122438). In the
cases of NK603 and MON 810 we found some sequence
divergence leading to different PCR efficiencies that result in
problems with the interpretation of quantification.
Although the quantification of a housekeeping gene is
recommended by some authors for exact quantification of the
target, there are some drawbacks, mainly because of additional
imprecision in the RT quantification, generated either by the
dilution of the DNA or by differences in the fluorescence
emission in the reports (1). In our case we prefer the conven-
tional spectrophotometrical DNA quantification and use the
amplification of invertase as positive control to approve the
The plasmid standards enable more flexibility and accuracy
in preparation but still show the same amplification efficiency
as complex samples in the system. The RSD of ct values is
constant and reproducible along the whole calibration interval,
and the genomic DNA samples from maize flour did not reveal
significant differences after a couple of freeze-thawing cycles.
Therefore, we conclude that the CTAB purification method is
well suited for generating sufficient amounts of DNA with
adequate quality. This validated method combines the potential
of the TaqMan probe method with the easy handling of a
plasmid calibration curve.
We thank Christine Lattenmayer and Sonja Preis for perfect
(1) Hera ´ndez, M.; Duplan, M. N.; Berthier, G.; Vaitilingom, M.;
Hauser, W.; Freyer, R.; Pla, M.; Bertheau, Y. Development and
comparison of four Rti-PCR systems for specific detection and
quantification of Zea mays L. J. Agric. Food Chem. 2004, 52,
(2) Vaı ¨tilingom, M.; Pijnenburg, H.; Gendre, F.; Brignon, P. Real-
time quantitative PCR detection of genetically modified Maxi-
mizer maize and Roundup Ready soybean in some representative
foods. J. Agric. Food Chem. 1999, 47, 5261-5266.
(3) James, C. Global status of commercialized transgenic crops.
ISAAA Briefs 2000, No. 23.
(4) Lipp, M.; Brodmann, O. IUPAC collaborative trial study of a
method to detect genetically modified soy beans and maize in
dried powder. J. AOAC Int. 1999, 82, 923-928.
(5) 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 1999,
10 (6), 385-389.
(6) Sanger, F.; Nickeln, S.; Coulson, A. DNA sequencing with chain
terminating inhibitors. Biotechnol. Bioeng. 1992, 24, 104-108.
(7) Rozen, S.; Skaletsky, H. J. Primer3 on the WWW for general
users and for biologist programmers. In Bioinformatics Methods
and Protocols: Methods in Molecular Biology; Krawetz, S.,
Misener, S., Eds.; Humana Press: Totowa, NJ, 2000; pp 365-
(8) Wegscheider, W.; Rohrer, Ch.; Neubo ¨ck, R. Manual Validata,
version 3.02; Feb 1999.
(9) Gardner, R. C.; Howarth, A. J.; Hahn, P.; Brown-Luedi, M.;
Shepherd, R. J.; Messing, J. The complete nucleotide sequence
of an infectious clone of cauliflower mosaic virus by M13mp7
shotgun sequencing. Nucleic Acids Res. 1981, 9 (12), 2871-
(10) Yanagisawa, S.; Izui, K. MNF1, a leaf tissue-specific DNA-
binding protein of maize, interacts with the cauliflower mosaic
virus 35S promoter as well as the C4 photosynthetic phospho-
enolpyruvate carboxylase gene promoter. Plant Mol. Biol. 1992,
19 (4), 545-553.
(11) Permingeat, H. R.; Reggiardo, M. I.; Vallejos, R. H. Detection
and quantification of transgenes in grains by multiplex and real-
time PCR. J. Agric. Food Chem. 2002, 50, 4431-4436.
(12) Holden, M. J.; Blasic, J. R.; Bussjaeger, L.; Kao, C.; Shokere,
L. A.; Kendall, D. C.; Freese, L.; Jenkins, G. R. Evaluation of
extraction methodologies for corn kernel (Zea mays) DNA for
detection of trace amounts of biotechnology-derived DNA. J.
Agric. Food Chem. 2003, 51, 2468-74.
(13) Tarverniers, I.; Windels, P.; Van Bockstaele, E.; De Loose, M.
Use of cloned DNA fragments for event specific quantification
of genetically modified organisms in pure and mixed food
products. Eur. Food Res. Technol. 2001, 213, 417-424.
(14) Trapman, S.; Catalani, P.; Conneely, P.; Contreras, M.; Corbisier,
P.; Gancberg, D.; Gioria, S.; Le Guern, L.; Linsinger, T.;
Schimmel, H. The certification of reference materials of dry-
mixed maize powder with different mass fractions of Bt-11
maize. EC Certification Report EUR 20985; 2003; ISBN 92-
Received for review September 13, 2005. Revised manuscript received
November 2, 2005. Accepted November 7, 2005. This work was kindly
sponsored by Agrana Maissta 1rkefabrik.