Food Control 16 (2005) 535–539

www.elsevier.com/locate/foodcont

A general multiplex-PCR assay for the general detection of

genetically modified soya and maize
V.T. Forte a, A. Di Pinto a,*
, C. Martino b, G.M. Tantillo a, G. Grasso c, F.P. Schena b

a
Facoltá di Medicina Veterinaria, Dipartimento Sanitá e Benessere degli Animali, Universotá degli Studi di Bari,
Provinciale per Casamassima, km 3, 70010 Valenzano––Bari, Italy
b
Apuliabiotech S.C.R.L., Provinciale per Casamassima, km 3, 70010 Valenzano––Bari, Italy
c
Tecnopolis C.S.A.T.A, Provinciale per Casamassima, km 3, 70010 Valenzano––Bari, Italy

Received 10 November 2003; received in revised form 22 May 2004; accepted 24 May 2004

Abstract

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The
European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or
food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling
requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important
GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya
or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically
modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving
and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified
soya and maize.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: GMO detection; Multiplex-PCR; Maize; Soybean

1. Introduction and/or utilize GMOs as food or food ingredients.

The use of genetically modified organisms (GOMs) as
food and in food products is becoming more and more
widespread. Genetically-engineered plants have been ap-
proved by several countries, such as US, Canada, Euro-
pean Union (EU), Switzerland, Australia, Argentina,
Brazil and Japan. The two most cultivated GMOs are
maize and soya, which represent the staple constituents
of many foods (Gachet, Martin, Vigneau, & Meyer,
1999).
The European Union has implemented a set of very
strict procedures for the approval to grow, import
*
Corresponding author. Tel.: +39 080 544 3970; fax: +39 080 544
3855.
E-mail address: a.dipinto@veterinaria.uniba.it (A. Di Pinto).
Throughout the whole legislative procedure, the EU
ensures that the European consumersÕ rights to be fully
informed are guaranteed. According to the European
regulation, specific labelling requirements must be ap-
plied to foodstuffs that are considered to be no longer
equivalent to an existing food ingredient (Reg. 1139/
98). In the case of GMO-food, these detectable differ-
ences can relate to the presence of newly introduced for-
eign DNA sequences or newly expressed proteins.
Therefore control of the labelling of such foodstuffs is
based on the detection of the foreign DNA sequences
born by the genetically-modified organisms.
Thus analytical methods for the detection of GMOs
are necessary in order to verify compliance with label-
ling requirements. The control and detection of such
foodstuffs is based on protein detection tests using

0956-7135/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2004.05.010
536 V.T. Forte et al. / Food Control 16 (2005) 535–539
antibodies like the ELISA-test (enzyme-linked immuno-
sorbent assay) and on polymerase chain reaction (PCR)
methods. Protein detection by immunoassay requires
the use of monoclonal antibodies raised against the pro-
tein encoded by the transgene (Lipp, Anklam, & Stave,
2000; Stave, 1999).
In the last years different PCR-based methods for the
specific detection of the most economically important
GMOs have been proposed. The specific sequences de-
tected are used to build the different GMOs and regulate
expression of transgenes, such as promoter 35S and the
terminator tNOS.
A qualitative screening PCR-based method to detect
most of the GMOs presently approved for marketing
was validated by assaying flour from Roundup Ready
soybeans and from BT 176 maize for the presence of
introduced DNA sequences (Lipp, Brodmann, Pietsch,
Pauwels, & Anklam, 1999). It is based on the detection
of the newly introduced genes, the so-called promoter
and terminator.
Currently a great deal of GMOs already approved or
under approval can be detected because they have share
the same DNA promoter and terminator sequences.
The method for analyzing a sample involves a first
amplification of specific soya or maize sequences from
plant DNA, necessary to discriminate between negative re-
sults and false negative results due to inhibition in the
amplification. The second step entails amplification of
GMO-specific sequences, represented by 35S promoter
and NOS terminator, to screen for the presence of trans-
genic material in samples. Then GMO-containing samples
are subjected to analysis of specific transgenes to determine
the strain of GMO present (Lin, Chiueh, & Shih, 2000).
The aim of this study was to develop a rapid, easy
and less time-consuming molecular method for screen-
ing GMOs. The authors describe a method based on
the multiplex-PCR (m-PCR) to identify simultaneously
the two plant genes, zeine and lectine, and the regulatory
sequences, 35S promoter and the NOS terminator, for
the detection of GMOs.
2. Materials and methods
2.1. Reference materials
In this study Roundup Ready GM-soybean (Mons-
anto, USA) with 0%, 0.1%, 0.5%, 1% and 2% GMO con-
tents, Bt 176 maize (Novartis/Ciba-Geigy, USA) with
0%, 0.1%, 0.5%, 1% and 2% GMO contents, Bt 11 maize
(Syngenta, USA) with 0%, 0.1%, 0.5%, 1% and 2%
GMO contents and MON 810 (Monsanto, USA) with
0%, 0.1%, 0.5%, 1% and 2% GMO contents were used
as negative and positive control. Reference materials
were obtained from Fluka Chemical (Switzerland) and
used as positive and negative controls.
2.2. DNA extraction
DNA extraction was carried out using wizard mag-
netic DNA purification System for Food (Promega,
Madison, WI, USA). 200 mg of each reference material
were added to 500 ll of Lysis Buffer A and 5 ll of Rnase
A. The mixture was obtained by vortexing and 250 ll of
Lysis Buffer B was then added. After incubation at room
temperature for 10 min, 750 ll of precipitation solution
was added and a vigorously blended. The mixture was
centrifuged for 10 min at 13.000g. Then the supernatant
was added to 50 ll of resuspended MagneSilä PMPs.
After vigorously vortexing, 0.8 vol isopropanol was
added. The tubes were mixed and incubated at room
temperature for 5 min with shaking. Then the tubes were
inserted into the MagneSphere Technology Magnetic
Separation Stand and left in place for 1 min. The liquid
phase was discarded leaving the tubes in the stand. The
tubes were remove from the stand and 250 ll of Lysis
Buffer B was added to the particles. The tubes were
mixed and replaced on MagneSphere Technology Mag-
netic Separation Stand. After 1 min incubation at room
temperature, the liquid phase was discarded. Then, 1 ml
of 70% ethanol wash solution was added and, after 1
min in the magnetic stand, the liquid phase was dis-
carded. This step was repeated for three times and in
the end the particles were dried at room temperature
for 15–30 min. 100 ll of nuclease-free water was added
to particles and the mixture obtained was mixed and
incubated at 65 °C for 5 min. The tube was inserted in
the MagneSphere Technology Magnetic Separation
Stand for 1 min and the DNA was collected by leaving
the tube in the stand and carefully transferring the liquid
into a new tube. The final volume was adjusted to 100 ll
by adding nuclease-free water. The DNA concentration
and the purity of the eluate were measured by absorb-
ance at 260 nm and by calculating the ratio of absorb-
ance at 260 nm to absorbance at 280 nm using a
spectrophotometer DU-600 (Beckman, Fullerton, CA).
The quality of extracted DNA was assessed by PCR
assay on chloroplast using universal primers for amplifi-
cation of three non-coding regions of chloroplast DNA
(Taberlet, Gielly, & Bouvet, 1992). The DNA eluted was
used as template in Multiplex PCR.
2.3. PCR primers
The sequences of oligonucleotide primers used in this
study are reported in Table 1. The primers were synthe-
sized by MWG Biotech (Milan, Italy).
2.4. Multiplex-PCR (m-PCR)
The m-PCR was carried out in touchdown using a
premix of 2X PCR reagent, AccuPrimeä SuperMix
II (Invitrogen, Japan) containing 40 mM Tris–HCl
 V.T. Forte et al. / Food Control 16 (2005) 535–539 537

Table 1
Oligonucleotide primers
Gene Orientation Sequence (5 0 –3 0 ) Accession number Amplicon (bp) Reference
0 0
LECTIN Upstream primer 5 –tgc cga agc aac caa aca tga tcc t–3 K00821 414 Brodmann et al. (1997)
Downstream primer 5 0 –tga tgg atc tga tag aat tga cgt t–3 0

ZEIN Upstream primer 5 0 –agt gcg acc cat att cca g–3 0 X07535 277 Studer et al. (1997)
Downstream primer 5 0 –gac att gtg gca tca tca ttt–3 0

NOS Upstream primer 5 0 –gaa tcc tgt tgc cgg tct tg–3 0 AY125353 180 Lipp et al. (1999)
Downstream primer 5 0 –tta tcc tag ttt gcg cgc ta–3 0

35S Upstream primer 5 0 –tgc ctc tgc cga cag tgg tc–3 0 AY192160 83 Trapmann et al. (2002)
Downstream primer 5 0 –aag acg tgg ttg gaa cgt ctt c–3 0

(pH 8.4), 100 mM KCl, 3 mM MgCl2 400 lM dGTP,
400 lM dATP, 400 lM dTTP, 400 lM dCTP, Accu-
Primeä Taq DNA Polymerase Thermostable Accu-
Primeä and protein stabilizers. PCR was performed in
a final volume of 50 ll containing 25 ll AccuPrimeä Su-
perMix II, 0.09 lM of each specific primer for endog-
enous genes, 0.15 lM of each specific primer for NOS
terminator and 35S promoter, 50 ng of each reference
template. MgCl2 was corrected to 2 mM. The primer
concentration for m-PCR was optimized by the determi-
nation of the minimum primer concentration.
The touchdown PCR was performed with an initial
denaturation step at 94 °C for 2 min followed by 15
cycles, which involved a denaturation step at 94 °C for
15 s, annealing at 65 °C for 30 s in the initial cycle
and at decreasing temperatures by 1 °C/cycle until a
temperature of 50 °C was reached in the subsequent
cycles. The extension step was performed at 68 °C for
1 min. After the touchdown program, 20 cycles at
94 °C for 15 s, 50 °C for 30 s, 68 for 1 min, followed
by a final extension at 72 °C for 7 min, were performed.
The m-PCR was programmed in a Mastercycler (Eppen-
dorf, Milan, Italy). The specificity of the amplified prod-
ucts was evaluated by sequencing performed by ABI
PRISM 3100 (Applied Biosystem, Rome, Italy).
method was able to minimize DNA degradation and en-
sured the isolation of pure and amplifiable DNA (data
not shown).
The m-PCR system applied to reference materials
showed only specific amplicons of the expected size.
The results related to Bt 176 maize (Fig. 1) and MON
810 maize standards with 0%, 0.1%, 0.5%, 1% and 2%
GMO contents showed the zein amplicons from 2% to
0% and 35S amplicons from 2% to 0.1%.
The m-PCR results referred to Roundup Ready GM-
soybean standards with 0%, 0.1%, 0.5%, 1% and 2%
GMO contents showed the lectin amplicons from
2% to 0% and NOS and 35S amplicons from 2% to
0.5% (Fig. 2). The Bt 11 maize standards with 0%,
0.1%, 0.5%, 1% and 2% GMO contents gave the zein
amplicons from 2% to 0% and 35S and NOS amplicons
from 2% to 0.5% (Fig. 3). The lectin, zein, NOS and 35S
amplicons were of 414, 217, 180 and 83 bp respectively.

2.5. Amplified products detection

PCR amplified products were analyzed by electro-

phoresis on 2.5% (w/v) agarose NA (Pharmacia, Upp-
sala, Sweden) gel in 1X TBE buffer containing 0.89 M
Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0 (USB,
Cleveland, OHIO) and visualized by ethidium bromide
staining and UV transilluminator. A Gene Rulerä 100
bp DNA Ladder Plus (MBI Fermentas, Vilnius, Lithua-
nia) consisting of DNA fragments ranging in size from
3000 to 100 bp, was used as marker. Fig. 1. Electrophoretic profile of m-PCR products: Lane 1: 100 pb
DNA Ladder; Lane 2: m-PCR products of Bt 176 2% GMO content;
Lane 3: m-PCR products of Bt 176 1% GMO content, Lane 4: m-PCR
3. Results
products of Bt 176 0.5% GMO content; Lane 5: m-PCR products of Bt
176 0.1% GMO content; Lane 6: m-PCR products of Bt 176 0% GMO
The quality of extracted DNA, assessed by PCR as- content; Lane 7: negative (no DNA) control. The arrow points to a
say on chloroplast DNA, indicated that the extraction weak band of maize Bt 176 0.1% 35S amplicon.
538 V.T. Forte et al. / Food Control 16 (2005) 535–539
Fig. 2. Electrophoretic profile of m-PCR products: Lane 1: 100 pb
DNA Ladder; Lane 2: m-PCR products of Roundup Ready GM-
soybean 2% GMO content; Lane 3: m-PCR products of Roundup
Ready GM-soybean 1% GMO content, Lane 4: m-PCR products of
Roundup Ready GM-soybean 0.5% GMO content; Lane 5: m-PCR
products of Roundup Ready GM-soybean 0.1% GMO content; Lane
6: m-PCR products of Roundup Ready GM-soybean 0% GMO
content; Lane 7: negative (no DNA) control.
Fig. 3. Electrophoretic profile of m-PCR products: Lane 1: 100 pb
DNA Ladder; Lane 2: m-PCR products of Bt 11 2% GMO content;
Lane 3: m-PCR products of Bt 11 1% GMO content, Lane 4: m-PCR
products of Bt 11 0.5% GMO content; Lane 5: m-PCR products of Bt
11 0.1% GMO content; Lane 6: m-PCR products of Bt 11 0% GMO
content; Lane 7: negative (no DNA) control.
The maize and soy 0% GMO contents results indicated
the presence of lectin and zein amplicons only. The tem-
plate-free negative control did not show the presence of
any amplicon. No false-positive results due to probable
contamination of laboratory equipment, were high-
lighted. The sequence analysis corresponded to the pub-
lished sequence of the tested reference materials.
These results, representative of ten experiments, did
not show variability and highlighted a high reproduci-
bility.
4. Discussion
The importance of the detection of genetically modi-
fied organisms (GMOs) in food is increasing dramati-
cally. The European Commission recently adopted an
important legislative package on GMOs which estab-
lishes a sound community system to trace and label
GMOs and to regulate the placement on the market
and labelling of food and feed products derived from
GMOs (Reg 258/97, Reg 1139/98, Reg 49/00, Reg 50/
00). The new legislation is intended to provide a trust-
worthy and environmentally safe approach to GMOs,
GM food and GM feed. The package consists of a pro-
posal to trace and label of GMOs and products produced
from GMOs and a proposal on regulating GM food and
feed. It requires that GMOs be traceable throughout the
chain from farm to table and provide consumers with
information by labelling all food and feed consisting
of, containing or produced from a GMO. Thus the Euro-
pean Union laws on labelling and tracing of GMOs have
created the need to detect different GMOs in large series
of samples and in many different sample types to enable
freedom of choice and ensure environmental safety.
An essential prerequisite for the application of food
labelling directives is the availability of analytical con-
trol methods (Anklam, 1999). PCR-related techniques
appear to be the methods of choice because of their high
sensitivity and specificity. PCR-based methods are based
on the stability of nucleic acid chains that can endure at
the usual treatments involved in food manufacturing.
The method developed for the detection of GMOs in-
volves a one step amplification of specific soya or maize
sequences from plant DNA to discriminate between neg-
ative results due to simple inhibition in the amplifica-
tion, and the amplification of GMO-specific sequences,
which include two transgenic sequences, parts of the
35S promoter and the NOS terminator, present in
around 95% of currently commercialised GMO plants
in EU. Traditionally, the detection of specific soya,
maize and GMO sequences were performed in different
amplification steps (Lipp et al., 2000).
The m-PCR assay discriminated the GMO very
quickly, reproducibly and in a cost-saving and less
time-consuming way. It is also a flexible assay because
it is carried out on the same tube in the same run. The
advantage of multiplex methods is that fewer reactions
are needed to test a sample for potential presence of
GMO-derived DNA. In particular, it may be of impor-
tance to exclude the analysis of a part of GMO since
qualitative and quantitative PCR are relatively expen-
sive. The optimised procedure represents a break-
 V.T. Forte et al. / Food Control 16 (2005) 535–539 539

Table 2
Results interpretation
LECTINA ZEINA 35S tNOS EVENT
+    No GMO containing 35S and tNOS
 +   No GMO containing 35S and tNOS
+ +   No GMO containing 35S and tNOS
  + + GMO different from maize and soya
  +  GMO different from maize and soya
   + GMO different from maize and soya
 + +  GMO Maize event and/or other event different from Maize BT 11, GA 21 Roundup Ready Corn
 +  + GMO Maize event and/or other event different from Maize BT 176, MON 810, T 25, BT 11, Starlink
 + + + GMO Maize event and/or other even different from soya
+  +  GMO Soya event and/or other event different from Soya Roundup Ready
+   + GMO Soya event and/or other event different from Soya Roundup Ready
+  + + GMO Soya event and/or other event different from maize

through screening method that applies genetics-based
testing to grains, food ingredients and processed food
products for fast, accurate results. The positive results
require further analysis to establish whether the maize
or soy is among the authorized GMO and to eventually
evaluate the amounts present. It may also be used to de-
tect the presence of unapproved GMOs different from
maize and soy according Italian laws (DPR 128/1999;
D.P.C.M. 04.08.2000), containing 35S promoter or
NOS terminator (Table 2). Further studies for the devel-
opment of methods for detection of GMOs in processed
food products, together with studies for the validation
of analytical methods are necessary to ensure consum-
ersÕ the safety and freedom of choice.
Further studies are necessary to evaluate the reliabil-
ity to processed ingredients and end products.
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