Multiplex GMO Screening: a Unique 4-Target Real-Time PCR

Multiplex GMO Screening: a Unique 4-Target Real-Time PCR
H.-H. Dörries, I. Remus, A. Grönewald, C. Grönewald, C. Harzman*, Berghof-Jäger
BIOTECON Diagnostics GmbH, Hermannswerder 17, 14473 Potsdam, Germany
*corresponding author: Christina Harzman, BIOTECON Diagnostics, CHarzman@bc-diagnostics.com
Phone: +49 (0)331 2300-282, Fax: +49 (0)331 2300-250
ABSTRACT
The ever-increasing number of commercialized GMOs forces laboratories to apply many different
methods of analysis. Screening methods help minimize analytical effort and pre-select for further tests
(e.g. GMO quantification). A unique real-time PCR kit (detection limit: 10 target copies/reaction) was
developed to detect the four most frequent target sequences used for GMO screening:
the 35S promoter of the cauliflower mosaic virus (CaMV), the NOS terminator of Agrobacterium
tumefaciens, the 35S promoter of the figwort mosaic virus (FMV) and the bar gene of Streptomyces
hygroscopicus.
INTRODUCTION
Among the molecular methods that are currently
available for analysis of genetically modified
organisms (GMO), real-time PCR is the gold
standard due to its specificity, sensitivity and
reliability as well as the possibility of quantification
[1-4]. One further advantage of DNA-based
methods is that DNA as a target molecule is more
robust with respect to degradation than for instance
proteins, which is an important advantage when
analyzing highly processed food samples.
Due to different specificity, there are a number of
different PCR methods for the detection of GMOs
[1, 5]. These include:
1. Screening assays that are specific for
genetic elements that are commonly used
in GMO development, such as gene
promoters or terminators. A positive
screening result does not necessarily imply
the presence of GMO-derived DNA. The
detected genetic elements might as well
occur naturally through the presence of the
host organisms like for example the
cauliflower mosaic virus (CaMV) [6].
1
2. Gene-specific assays, e. g. for the detection
of genes transferring herbicide resistance
or pest resistance to the host crop.
Elements like these are also often used for
screening and occur naturally. However,
slightly modified versions of the genes are
often used in GMOs [7].
3. The third assay category comprises PCR
methods specific for a junction between
different elements within the GMO
sequence.
4. Event-specific GMO detection methods
are the most specific ones. They span a
junction between the wildtype plant DNA
and the foreign GMO sequences. This is
achieved by designing one primer specific
for the foreign gene construct and the other
primer specific for the flanking genomic
sequence. Since the only difference
between several events containing the
same construct is its location within the
host genome, PCR is the only method for
identification and quantification of specific
events.

As there are currently more than 140 GM crops
worldwide that have been approved for cultivation
and human consumption, multiplex assays and
effective test schemes utilizing the different
methods mentioned above in a step-wise analysis
have been established [8-9]. The initial step in
routine GMO analysis is a preliminary screening
assay to gather information in order to minimize the
number of further identification and quantification
tests [9]. As food processing often results in
degraded DNA, assays for the determination of the
overall quality of plant DNA in general are often
used. Additionally, real-time PCR assays should
always include an internal amplification control
(IAC) for identification of false-negative results due
to PCR inhibition.
Multiplex methods for the dual detection of the
CaMV 35S promoter and the terminator of the
nopaline synthase gene of Agrobacterium
tumefaciens (T-NOS or NOS) have been validated
and are commonly used for GMO screening [10-
11]. To thoroughly detect as many genetically
modified crops as possible an expansion of existing
screening methods is required. For this purpose,
assays that are specific for other elements like e. g.
the 35S promoter of the figwort mosaic virus
(FMV) can be included [12]. The FMV promoter is
part of the transgene sequence of new approved
events like soya MON89788 (Roundup Ready 2
Yield) and sugar beet H7-1 (Roundup Ready).
The application of screening methods makes it also
possible to detect non-approved GMOs for which
no specific assays, reference material or even
genetic sequences are available. Recently, a
strategy was employed to detect unknown GMOs
using arrays with 25 basepair probes covering both
strands of a set of 235 vectors (2 million basepairs)
without prior knowledge of the used transformation
vectors [14].
This technical paper summarizes the development
of the foodproof ® GMO Screening Kit (35S, NOS,
bar, FMV), 5’Nuclease. This is the first commercial
kit offering multiplex real-time PCR for the
screening of four GMO targets at once. The
detection system contains an assay for the
2
simultaneous detection of four important genetic
elements and a second assay for control of DNA
integrity and inhibition control. The GMO assay is
specific for the CaMV 35S promoter, the NOS
terminator of Agrobacterium tumefaciens, the bar
gene of the soil bacterium Streptomyces
hygroscopicus and the FMV 35S promoter. An
additional control assay detects plant DNA and the
internal amplification control (IAC) for the
identification of false-negative results.
MATERIALS AND METHODS
Sample Material and DNA Isolation. 26 bacterial,
25 yeast and 13 mold species were used for the
testing of the assay specificity. They were obtained
from the DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen), the ATCC
(American Type Culture Collection), the NCTC
(National Collection of Type Cultures), the MUCL
(Mycotheque de l'Universite catholique de
Louvain) or the CBS (Centraalbureau voor
Schimmelcultures). For further specificity testing,
DNA was extracted from 41 non-genetically
modified plants. DNA isolation was conducted with
the foodproof ® ShortPrep II Kit for bacteria, with
the foodproof ® StarPrep Four Kit for yeasts or
molds and with the foodproof ® GMO Sample
Preparation Kit (all three kits: BIOTECON
Diagnostics GmbH, Potsdam, Germany) for plants.
50 different food products out of different groups
(e.g. maize, tacos, frozen food, popcorn, maize
starch, muesli/cereal bars, tofu, meat substitute,
soya beans, soya drinks, lectithin) were processed
with the foodproof ® GMO Sample Preparation Kit
for the applicability study. 5 µl of the unspiked
sample preparations made with the foodproof ®
GMO Sample Preparation Kit were used for PCR.
Afterwards, samples were artificially contaminated
by spiking 3 µl of the sample preparations with 2 µl
of a mix containing purified and quantified DNA
(200 target copies/µl) from the following GMO
reference material: MON 89788 (AOCS 0906-B),
Bt176 (IRMM-411) and EH92-527-1 (IRMM-421).
Further GMO reference materials were obtained
from Sigma-Aldrich or AOCS (American Oil
Chemists' Society): GTS 40-3-2 (IRMM-410),
MON 89788 (AOCS 0906-B), A2704-12 (AOCS
0707-B), Bt11 (IRMM-412), Bt176 (IRMM-411),

CBH-351 (Sigma-Aldrich, 69407), GA 21 (Sigma-
Aldrich, 69407), MIR604 (IRMM-423), MON 810
(IRMM-413), MON 863 (IRMM-416), NK 603
(Sigma-Aldrich, 69407), T25 (AOCS 0306-H),
TC1507 (IRMM-418), GS40/90 (Sigma-Aldrich,
55231), GT200/GT73 (Sigma-Aldrich, 55231),
MS8xRf3 (Sigma-Aldrich, 55231), Oxy235
(Sigma-Aldrich, 55231), T45 (AOCS 0208-A),
LLRice62 (AOCS 0707-B), H7-1 (IRMM-419),
EH92-527-1 (IRMM-421) and LLCotton25 (AOCS
0306-E).
LOD (limit of detection) determination as well as
the spiking of prepared food samples were carried
out with protein-free and RNA-free DNA from
GMO reference material, quantified
photometrically with a NanoPhotometer (Implen
GmbH, München, Germany). The number of the
target sequence copies was calculated by assuming
that the haploid genome sizes of soya, maize, sugar
beet and potato are 1.1 pg, 2.7 pg, 0.9 pg and 0.9
pg, respectively [15].
Real-time PCR. The foodproof ® GMO Screening
Kit (35S, NOS, bar, FMV) for the detection of
genetically modified organisms consists of two
real-time PCR systems based on hydrolysis (5’nuclease)
probes or TaqMan ® technology. Each
system consists of a premade master mix
(35S/NOS/bar/FMV or Plant Gene Master Mix)
containing primers, probes and all further necessary
reagents, an enzyme solution and an internal
amplification control or dye solution. The enzyme
solution contains blocked “HotStart” Taq DNA
polymerase and the enzyme uracil-DNA
glycosylase (UDG) for decontamination and thus
prevention of false-positive results. Furthermore, a
positive control DNA is also provided with the kit
for all five assays.
Four PCR assays specific for screening elements
commonly used in GMOs were designed for the
35S/NOS/bar/FMV Master Mix. The amplicons of
the CaMV 35S promoter, the 3'-untranslated region
of the nopaline synthase gene of Agrobacterium
tumefaciens (NOS terminator), the bar resistance
gene (phosphinothricin N-acetyltransferase,
transferring herbicide resistance) of the soil
3
bacterium Streptomyces hygroscopicus and the
FMV 35S promoter were simultaneously detected
using specific hydrolysis probes and visualized by
monitoring the resulting fluorescent signal of the
four used reporter fluorescence dyes. The reporter
dyes attached to the 5’-end of the GMO-specific
probes were FAM (6-carboxy-fluorescein) for the
35S assay, HEX (6-carboxy-2',4,4',5',7,7'hexachlorofluorescein)
for the NOS assay, ROX (5carboxy-X-rhodamine)
for the bar assay and Cy5
for the FMV assay. At the 3’end all probes were
labeled with a dark quencher.
The Plant Gene Master Mix contains specific
oligonucleotides for the detection of plant-derived
DNA and a homologous internal amplification
control (IAC). The IAC, which contains the
fragment of the plant target with a modified probe
binding sequence, is amplified by the primers of the
plant assay but the amplicon is detected with a
different probe. For the detection of the plant gene
and the IAC the probes were labeled with FAM and
HEX at the 5’-end and with a dark quencher at the
3’-end, respectively.
PCR was set up according to the supplier’s
instructions. For each reaction 18 µl
35S/NOS/bar/FMV Master Mix or Plant Gene
Master Mix were mixed with 1 µl Enzyme Solution
and 1 µl Dye Solution (35S/NOS/bar/FMV system)
or 1 µl Internal Amplification Control (Plant Gene
system). 20 µl of either master mix were then
transferred into the corresponding well of a 96multiwell
PCR plate and 5 µl PCR grade water
(negative control), 5 µl positive control or 5 µl of
food sample DNA were added, respectively.
PCR conditions were the following:
1. Pre-incubation and initial denaturation:
4 min at 37 °C
10 min at 95 °C
2. Amplification (50 cycles)
5 s at 95 °C
60 s at 60 °C (signal acquisition)
Fluorescence signals were measured after the
second step of the amplification program.

Real-time PCR experiments were run on the
LightCycler 480 instrument (Roche Diagnostics
GmbH, Mannheim, Germany) or the Mx3005P
QPCR System (Agilent Technologies, Waldbronn,
Germany). Each experiment consisted of the
samples to be analyzed as well as one positive and
one negative control each for both master mixes
(35S/NOS/bar/FMV and Plant Gene).
RESULTS AND DISCUSSION
Specificity. Primer and probe concentrations,
reaction conditions and PCR profile were adjusted
in order to optimize the performance of both
multiplex assays. The expected results were
achieved for all tested reference materials with the
35S/NOS/bar/FMV Master Mix and the Plant Gene
Master Mix after testing with the LightCycler 480
instrument (Table 1). Only events containing the
respective screening target were positive with the
corresponding assay and all events were positive
with the plant-specific assay. It can be predicted
from databases that the 35S/NOS/bar/FMV Master
Mix detects almost all of the GMO events for
which information on the inserted genetic elements
is available [13]. Only two soybean events
(DP305423 and DP356043) and one canola event
(GT200) lacking the target sequences cannot be
detected with this four-target screening method.
Conclusions regarding the identity of a GMO can
be made based on the combination of screening
targets present or absent in a sample. The detection
of a screening element in a sample can thus narrow
the group of possible events present. A soybean
sample with e. g. a positive signal for the FMV 35S
promoter should in a next step be checked for the
presence of the event MON 89788 (Roundup
Ready2Yield).
The identification of GMO events can be hampered
by the tendency that more transgenic varieties have
several (pyramided) traits incorporated [16].
Distinguishing stacked events produced by
hybridization from a mixture of the parent lines by
event-specific assays could not be achieved.
Besides, an overestimation of the relative GMO
level in a sample containing stacked event is
possible [17]. With regard to this problem, an
4
enhanced multiplex PCR technique for eventspecific
detection of maize event Bt11 x GA21 was
recently described [18]. For GMO analysis more
and more new assays are developed for the
simultaneous detection and identification of
multiple target sequences [19]. The use of multiplex
PCR assays GMO detection and identification has
been described previously [20-26]. Microarray
technology can potentially play an important role in
the future through the parallel detection of multiple
GMO-related sequences per sample [27-28].
Table 1: Real-time PCR results with certified reference
material of different GMO events
Reference
PCR assay
material 35S NOS bar FMV Plant
Soya
GTS 40-3-2 + + - - +
MON 89788 - - - + +
A2704-12 + - - - +
Maize
Bt11 + + - - +
Bt176 + - + - +
CBH-351 + + + - +
GA 21 - + - - +
MIR604 - + - - +
MON 810 + - - - +
MON 863 + + - - +
NK 603 + + - - +
T25 + - - - +
TC1507 + - - - +
Canola
GS40/90 + - - - +
GT200/GT73 - - - + +
MS8 x Rf3 - + + - +
Oxy235 + + - - +
T45 + - - - +
Rice
LLRice62 + - + - +
Sugar beet
H7-1 - - - + +
Potato
EH92-527-1 - + - - +
Cotton
LLCotton25 + - + - +
(+) positive result with specific PCR assay; (-) negative
result with specific PCR assay
The method’s exclusivity was tested with nontarget
DNA from different bacteria species, yeasts,
molds and non-genetically modified plants (see

Table 2). No amplification of any DNA sample
with the 35S/NOS/bar/FMV Master Mix was
observed. PCR inhibition was precluded for the
bacterial DNA samples by the amplification of the
internal amplification control.
All plant samples could be amplified with the Plant
Gene Master Mix verifying that the integrity of the
target DNA was not influenced by the DNA
isolation and purification process.
Sensitivity. The lowest quantity or concentration
that can be reliably detected with an acceptance
criterion is defined as the limit of detection (LOD).
LOD determination of the GMO-specific assays
was done with quantified and purified DNA of the
events MON 89788, H7-1, EH92-527-1, MON 810,
Bt 176 and GA 21. For each target sequence a serial
10-fold dilution of known concentrations (1,000 to
1 copies/reaction) was analyzed in 15 replicates.
The number of genome equivalents (Nge) per µl was
calculated according to the following equation:
Nge/µl = DNA-concentration [ng/µl] x 1000/haploid
genome [pg]. All four GMO target assays had an
absolute LOD of 10 initial template copies per
reaction (Table 3).
The actual amount of genome equivalents in a PCR
sample ranges from approximately 18,500 (50 ng
sample DNA) to 74,000 (200 ng sample DNA) if a
mass of 2.7 pg for an unreplicated haploid maize
genome (C value) was assumed [15]. Therefore, a
relative concentration of 0.05 % GMO genomes in
a sample (9.3 or 37.0 genome equivalents of the
GMO) was detectable with the GMO targeting
assays. Thus, the system can reliably detect the
relative GMO content to monitor e.g. the European
0.9 % labeling system. Previous studies of event
specific real-time PCR assays also had a limit of
detection of 10 to 20 initial template copies per
reaction [29-30].
The Cp value differences that were detected with
the maize events MON 810 and Bt 176 were caused
by differences in the reference material used for
DNA extraction. 20 genome equivalents resulted in
a Cp value of 24.50 for MON 810 and 31.96 for Bt
176 with the Plant Gene Master Mix. It was shown
5
by gel electrophoresis that the DNA extracted from
the Bt 176 flour was more degraded than the DNA
from the MON 810 flour (data not shown). In
general, DNA derived from flour is more processed
and of lower quality than the reference material
which was delivered as DNA, because much of the
DNA is degraded [29]. For protein preparations
made from soya and for processed tomato products
the available target sequences range from 100 to
400 bp [31], which is why assays with as small as
possible amplicon size are demanded. For this
purpose locked nucleic acid (LNA) 5’-nuclease
probes could be used to reduce the size of the
amplified fragments [32].
Robustness. The kit performance was tested with
50 different food products in order to simulate a
sample matrix more relevant to the situation in the
food industry. No positive results were detected for
the unspiked food samples with the
35S/NOS/bar/FMV Master Mix (data not shown).
In the food samples spiked with 400 copies/reaction
of all four target sequences the parallel detection of
all four sequences was possible. The single assays
did not influence each other.
PCR assays might be influenced by substances like
polysaccharides, phenolic compounds, proteins and
other secondary plant metabolites which are coextracted
from plant and food samples [33-34].
PCR inhibition through processed food with high
lipid content or food subjected to intense thermal
treatments was sometimes observed [35]. In case of
the presented assay, the results show that a high
amount of non-target DNA from different food
matrices had no negative influence on the target
sequence amplification.
Additional experiments carried out on the Mx3005p
instrument showed that the detection of 10
copies/reaction was possible (data not shown). Thus
the compatibility of the method with real-time
instruments other than the LightCycler 480 was
proven.
CONCLUSION
The foodproof® GMO Screening Kit (35S, NOS,
bar, FMV) allows the qualitative multiplex

Table 2: Results of the specificity testing with non-target DNA from bacterial species, yeasts, molds and non-genetically modified plants
Species (collection no.) 35S/NOS/bar/ Cp Cp IAC Plant 35S/NOS/bar/ Cp Cp IAC
FMV detection* Plant
FMV detection* Plant
Bacillus firmus (DSM 12) - - 34.89 Apple - 33.11 33.74
Bacillus stearothermophilus (DSM 456) - - 34.61 Banana - 22.59 -
Buttiauxella agrestis (DSM 4586) - - 34.67 Pear - 29.79 32.07
Cedecea davisae (DSM 4568) - - 43.72 Cauliflower - 23.18 -
Citrobacter koseri (DSM 4595) - - 34.73 Bean (green) - 32.55 33.67
Clostridium perfringens (DSM 12709) - - 34.48 Bean (red) - 34.99 34.52
Enterobacter aerogenes (DSM 30053) - - 34.83 Bean (white) - 22.73 -
Enterobacter amnigenus (DSM 4486) - - 34.46 cashew nut - 24.66 -
Enterobacter intermedius (DSM 4581) - - 35.38 Curcuma - 25.69 -
Cronobacter sakazakii (DSM 4485) - - 35.04 Chicory - 23.30 -
Ewingella americana (DSM 4580) - - 35.09 Spelt - 22.68 -
Erwinia carotovora (DSM 30168) - - 35.20 Pea - 20.82 -
Erwinia chrysanthemi (DSM 4610) - - 35.07 Strawberry - 24.57 -
Escherichia blattae (NCTC 12127) - - 35.58 Peanut - 25.09 -
Escherichia coli (NCTC 12790) - - 35.66 Oats - 23.97 -
Escherichia hermanii (DSM 4560) - - 34.69 Hazelnut - 21.35 -
Escherichia vulneris (DSM 4564) - - 35.11 Sorghum - 23.13 -
Hafnia alvei (DSM 30163) - - 41.89 Honeydew melon - 26.65 -
Klebsiella oxytoca (DSM 5175) - - 34.20 Potato - 22.76 -
Klebsiella planticola (DSM 4617) - - 34.9 Coco - 25.56 -
Klebsiella terrigena (DSM 2687) - - 34.81 Pumpkin - 23.92 -
Kluyvera ascorbata (DSM 4611) - - 35.38 Linseed - 28.08 29.89
Kluyvera cryocrescens (DSM 4588) - - 35.24 Lense - 21.35 -
Leclercia adecarboxylata (SM 5077) - - 34.77 Almond - 20.68 -
Moellerella wisconsensis (DSM 5076) - - 35.48 Nutmeg - 28.66 29.52
Plesiomonas shigelloides (DSM 8224) - - 35.04 Para nut - 30.86 -
Candida albicans (ATCC 10231) - - 35.60 Pecan - 25.13 -
Candida inconspicua (MUCL 27868) - - 35.30 Parsley - 30.55 32.82
Candida tropicalis (ATCC 20247) - - 35.03 Plum - 32.63 33.81
Clavispora lusitaniae (DSM 70102) - - 35.55 Pistachio - 20.73 -
Cryptococcus albidus (DSM 70197) - - 35.07 Rice - 26.58 -
Debaryomyces etchelsii (ATCC 20126) - - 35.08 Rye - 21.38 -
Dekkera bruxellensis (DSM 3429) - - 34.82 Sesame - 21.10 -
Endomyces fibuliger (DSM 70554) - - 34.71 Sunflower - 22.84 -
Filobasidium capsuligenum (DSM 70253) - - 35.07 Soya - 22.21 -
Guilliermondella selenospora (DSM 3431) - - 34.90 Spinach - 21.60 -
Hanseniaspora guilliermondii (DSM 70285) - - 35.33 Tobacco - 22.29 -
Hyphopichia burtonii (DSM 3505) - - 35.90 Tomato - 29.27 30.99
Issatchenkia orientalis (CBS 5147) - - 34.93 Tomato puree - 33.86 34.01
Kluyveromyces marxianus (DSM 70106) - - 35.08 Walnut - 21.20 -
Lachancea thermotolerans (CBS 6340) - - 35.51 Wheat - 22.19 -
Lodderomyces elongisporus (DSM 70320) - - 34.82
Metschnikowia pulcherrima (DSM 70321) - - 34.79
Pichia anomala (DSM 70783), - - 35.69
Rhodotorula minuta (DSM 70408) - - 34.92
Saccharomyces bayanus (DSM 70412) - - 35.34
Saccharomyces cerevisiae (ATCC 9763) - - 34.72
Schizosaccharomyces pombe (DSM 70844) - - 34.95
Torulaspora delbrueckii (CBS 5636) - - 34.79
Yarrowia lipolytica (ATCC 20225) - - 34.87
Zygosaccharomyces rouxii (DSM 70835) - - 35.66
Absidia corymbifera (DSM 1144) - - 34.93
Alternaria citri (CBS 106.27) - - 35.57
Aspergillus fumigates (DSM 819) - - 34.77
Aspergillus penicillioides (DSM 1623) - - 35.97
Claviceps purpurea (DSM 714) - - 34.46
Diaporthe citri (DSM 1159) - - 35.12
Eupenicillium abidjanum (DSM 2207) - - 34.98
Fusarium culmorum (DSM 62184) - - 34.26
Monilia fructigena (DSM 2678) - - 35.03
Mucor racemosus (CBS 225.37) - - 35.52
Neurospora crassa (DSM 1129) - - 34.93
Penicillium camembertii (DSM 1995) - - 35.27
Wallemia sebi (DSM 5329) - - 35.04
(-) negative with specific PCR assay; Cp = crossing point; IAC = internal amplification control
*all tested organisms were negative in all four fluorescence channels with the 35S/NOS/bar/FMV Master Mix
6

screening for the presence of GMOs with a
detection limit of 10 target DNA copies per PCR
reaction, which is well below the European
regulations requirements. The compatibility of this
method was proven on real-time instruments the
LightCycler 480 (Roche Diagnostics GmbH,
Mannheim, Germany) and the Mx3005P QPCR
System (Agilent Technologies, Waldbronn,
Germany). The foodproof® GMO Screening Kit
provides a basis for a rapid, sensitive and reliable
screening of food and feed for the most frequently
used genetic elements present in GM crops.
BIOTECON DIAGNOSTICS
COMPANY INFORMATION
BIOTECON Diagnostics is a biotechnology
company that focuses on development, production
and marketing of PCR-based rapid detection
systems. We offer solutions for the detection of
pathogens and GMOs for the food and beverage
industry as well as for producers of pharmaceuticals
and cosmetics. For more information about the
foodproof® GMO Screening Kit and to learn about
the foodproof GMO Soya or Maize Quantification
Kits also offered by BIOTECON Diagnostics,
contact the author or visit www.bc-diagnostics.com.
REFERENCES
1. Anklam E, Gadani F, Heinze P, Pijnenburg H, Van
den Eede G (2002) Eur Food Res Technol 214: 3–26
2. Alexander T W, Reuter T, Aulrich K, Sharma R,
Okine E K, Dixon W T, McAllister T A (2007)
Anim Feed Sci Technol 133:31-62
3. Miraglia M, Berdal K G, Brera C, Corbisier P,
Holst-Jensen A, Kok E J, Marvin H J P, Schimmel
H, Rentsch J, van Rie J P P F, Zagon, J (2004) Food
Chem Toxicol 42:1157-1180
4. Community Reference Laboratory, GM Food and
Feed, Ispra, http://gmocrl.jrc.ec.europa.eu/statusofdoss.htm
5. Holst-Jensen A, Rønning S B, Løvseth A and Berdal
K G (2003) Anal Bioanal Chem 375:985–993
6. Wolf C, Scherzinger M, Wurz A, Pauli U, Hübner P,
Lüthy J (2000) Eur Food Res Technol 210:367-372
7. Marmiroli N, Maestri E, Gullì M, Malcevschi A,
Peano C, Bordoni R, de Bellis G (2008) Anal
Bioanal Chem 392:369-384
8. James C (2008) ISAAA Brief 39
9. Michelini E, Simoni P, Cevenini L, Mezzanotte L,
Roda A (2008) Anal Bioanal Chem 392:355-367
7
10. Waiblinger H U, Ernst B, Anderson A, Pietsch K
(2008) Eur J Food Res Technol 226:1221-1228
11. Technische Regel BVL L 00.00-122 (2008)
12. Waiblinger H U, Boernsen B, Pietsch K (2008) Deut
Lebensm-Rundsch 104:261-264
13. AGBIOS, http://www.agbios.com/main.php
14. Tengs T, Kristoffersen A, Berdal K, Thorstensen T,
Butenko M, Nesvold H, Holst-Jensen A (2007)
BMC Biotechnol 7:91
15. Bennett M D, Leitch I J (1997) Ann Bot 80:169-196
16. Halpin C (2005) Plant Biotechnol 3:141-155
17. Akiyama H, Watanabe T, Wakabayashi K, Nakade
S, Yasui S, Sakata K, Chiba R, Spiegelhalter F, Hino
A, Maitani T (2005) Anal Chem 77:7421-7428
18. Xu W, Yuan Y, Luo Y, Bai W, Zhang C, Huang K
(2009) J Agric Food Chem 57:395-402
19. Chaouachi M, Chupeau G, Berard A, McKhann H,
Romaniuk M, Giancola S, Laval V, Bertheau Y,
Brunel D (2008) ) J Agric Food Chem 56:11596-
11606
20. Matsuoka T, Kuribara H, Akiyama H, Miura H,
Goda Y, Kusakabe Y, Isshiki K, Toyoda M and
Hino A (2001) J Food Hyg Soc Jpn 42:24-32
21. James D, Schmidt A-M, Wall E, Green M and Masri
S (2003) J Agric Food Chem 51:5829–5834
22. Shrestha H K, Hwu K-K, Wang S-J, Liu L-F and
Chang M-C (2008) J Agric Food Chem 56:8962-
8968
23. Onishi M, Matsuoka T, Kodama T, Kashiwaba K,
Futo S, Akiyama H, Maitani T, Furui S, Oguchi T,
Hino A (2005) J Agric Food Chem 53:9713-9721
24. Germini A, Zanetti A, Salati C, Rossi S, Forre C,
Schmid S, Marchelli R (2004) J Agric Food Chem
52:3275-3280
25. Hernandez M, Rodriguez-Lazaro D, Zhang D,
Esteve T, Pla M, Prat S (2005) J Agric Food Chem
53:3333-3337
26. Gaudron T, Peters C, Boland E, Steinmetz A, Moris
G (2009) Eur Food Res Technol 229:295-305
27. Xu J, Miao H, Wu H, Huang W, Tang R, Qiu M,
Wen J, Zhu S, Li Y (2006) Biosens Bioelectron
22:71-77
28. Leimanis S, Hernández M, Fernández S, Boyer F,
Burns M, Bruderer S, Glouden T, Harris N,
Kaeppeli O, Philipp P, Pla M, Puigdomènech P,
Vaitilingom M, Bertheau Y, Remacle J (2006) Plant
Mol Biol 61:123-139
29. Rønning S B, Vaïtilingom M, Berdal K G and Holst-
Jensen A (2003) Eur Food Res Technol 216:347–
354
30. Huang C-C, Pan T-M (2005) J Agric Food Chem
53:3833-3839

31. Hemmer W (1997) Foods derived from genetically
modified organisms and detection methods. BATS-
Report 2/1997, Agency for Biosafety Research and
Assessment of Technology Impacts of the Swiss
Priority Program Biotechnology of the Swiss
National Science Foundation, Basel, Switzerland
32. Salvi S, D’Orso F, Morelli G (2008) J Agric Food
Chem 56:4320-4327
33. Holden M J, Blasic J R, Bussjaeger L, Kao C,
Shokere L A, Kendall, D C (2003) J Agric Food
Chem 51:2468-2474
34. Wilson I G (1997) Appl Environ Microbiol 63:3741-
3751
35. Cazzola M L and Petruccelli S (2006) Electronic J
Biotechnol 9:320-325
8

Table 3: Amplification data for the determination of the absolute LOD of the 35S, NOS, bar and FMV assays and for the sensitivity of the plant assay
35S/NOS/bar/FMV Master Mix
template
copies per
reaction
Soya MON 89788
(FMV-promoter)
Rate of Mean Cp
recovery values (CV)
Sugar beet H7-1
(FMV-promoter)
Rate of Mean Cp
recovery values (CV)
Potato EH92-527-1
(NOS-terminator)
Rate of Mean Cp
recovery values (CV)
Maize MON 810
(35S-promoter)
Rate of Mean Cp
recovery values (CV)
Maize Bt 176
(35S-promoter/bar gene)
Rate of
Mean Cp values (CV)
recovery
Maize GA 21
(NOS-terminator)
Rate of Mean Cp values
recovery (CV)
1000 15/15 29.03 (0.33) 15/15 29.91 (0.93) 15/15 29.78 (0.56) 15/15 28.97 (4.73) 15/15; 15/15 29.79 (2.24); 30.93 (1.62) 15/15 29.53 (0.54)
100 15/15 32.14 (0.56) 15/15 32.89 (0.70) 15/15 32.83 (0.80) 15/15 31.70 (3.61) 15/15; 15/15 32.59 (1.59); 33.68 (1.37) 15/15 33.10 (1.59)
10 15/15 34.54 (1.77) 15/15 34.82 (2.03) 15/15 35.15 (1.66) 15/15 33.85 (0.35) 15/15; 15/15 34.69 (1.73); 35.57 (1.95) 15/15 35.51 (2.13)
1 6/15 35.47 (2.73) 7/15 35.18 (0.73) 12/15 36.18 (3.45) 8/15 35.25 (1.82) 7/15; 7/15 36.07 (1.25); 37.19 (0.74) 3/15 36.59 (0.93)
Soya MON 89788 Sugar beet H7-1
Plant Master Mix
Potato EH92-527-1 Maize MON 810 Maize Bt 176 Maize GA 21
Nge per Mean Cp Nge per Mean Cp Nge per Mean Cp Nge per Mean Cp Nge per Mean Cp Nge per Mean Cp
reaction values (CV) reaction values (CV) reaction values (CV) reaction values (CV) reaction values (CV) reaction values (CV)
1000 19.74 (0.46) 1000 21.72 (1.94) 1000 20.92 (0.50) 20000 14.08 (0.40) 20000 21.83 (1.26) 50000 15.75 (2.10)
100 23.14 (0.36) 100 25.04 (1.82) 100 24.37 (1.93) 2000 17.59 (1.30) 2000 25.20 (0.95) 5000 19.50 (3.40)
10 26.62 (0.44) 10 28.17 (1.01) 10 27.56 (0.82) 200 21.11 (1.71) 200 28.66 (1.41) 500 23.07 (3.42)
1 29.94 (0.54) 1 31.54 (1.29) 1 30.92 (0.66) 20 24.50 (1.02) 20 31.96 (1.29) 50 26.36 (2.24)
Cp = crossing point; CV = coefficient of variation; Nge = genome equivalents
9