Detection of Genetically Modified Maize and Soybean Food Products in the
The objective of this study was to survey for the genetically modified maize and soybean food products in the Jordanian market. The study was designed to extract genomic DNA of maize and soybean products by cetyltrimethylammonium bromide (CTAB) method and to identify specific genes for maize and soybean, expression control specific genes 35S promoter and NOS terminator by polymerase chain reaction analysis. For confirmation test of the genetically modified maize and soybean food products, nested polymerase chain reaction experiments were performed using internal primers for the detection of the E35S promoter and the hsp70 exon1/intron1 region of maize MON810 and Cp4 EPSPS gene of soybean. Three out of 19 maize food products were identified as carrying amplified DNA fragments of 35S promoter region, the nested PCR test confirmed the presence of MON810 event. One of three soybean food products was identified as carrier DNA fragments of 35S promoter region, Cp4 EPSPS event was not detected.
Received: July 19, 2010;
Accepted: September 09, 2010;
Published: October 21, 2010
The introduction of Genetically Modified Organisms (GMOs), also called Living
Modified Organisms (LMOs) and their products have made a great contribution
to the worlds economic development (Al-Hmoud et
al., 2008; Borlaug, 2000; Raveendar
and Ignacimuthu, 2010; Vain, 2006). Currently transgenic
maize and soybean are worldwide major crops with vital agronomical interests,
at present there are several GM products of these crops available in markets,
e.g., Roundup ReadyTM soy and MON810 Yield Gard Corn (Greiner
and Konietzny, 2008; Ujhelyi et al., 2008).
However, there are rising international concerns about possible potential risks
of GM products, this is because of the results of various studies which reported
possible harmful effect might be caused from the use of GMO or their products
(Konig et al., 2004; De
Vendomois et al., 2009; Seralini et al.,
2007, 2009). As a result of possible risks which
might be implicated by GMOs, increasing number of countries has adopted labeling
policies for GMOs and their products. Nowadays, countries with enforced labeling
policies include Australia, China, the European Union, New Zealand, Japan, Russia,
Saudi Arabia, South Korea, Switzerland and Taiwan and there are more countries
planning to introduce labeling policies (Gruere and Rao,
2007). Consequently studies have been carried out in several countries to
ascertain the presence and type of GM events of GM products. Type of genetic
modification of GM soybean and maize products in Egyptian market were identified
by Polymerase Chain Reaction (PCR), the study revealed 20% of 40 investigated
soy samples contained Roundup Ready soybean; 15% of 40 maize samples tested
positive for Bt176 and 12.5% positive for Bt11 maize (El-Sanhoty
et al., 2002). Polymerase-chain-reaction-based methods were used
to detect and survey genetically modified soy (Roundup-ReadyTM soy) and maize
(Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 LibertyR Link
maize) sold commercially in Brazil, the investigation showed the number of food
products containing genetically modified soy in a proportion above 1.0% on the
ingredient level, the threshold for labeling according to Brazilian legislation,
increased from 11% in 2000 to 36% in 2005 (Greiner and Konietzny,
2008; Greiner et al., 2005).
The objective of this study was to determine qualitatively by validated PCR methods the occurrence of products derived from genetically modified maize and soybean in foods sold commercially in Jordan. Maize and soybean were chosen because these are the major transgenic crops grown world-wide.
MATERIALS AND METHODS
Maize and soybean food products: Twenty two commercial food products,
19 of maize origin and three of soybean origin (Table 1),
were obtained during the period from January 2010 and June 2010 from Food and
Drug Testing Administration, the Royal Scientific Society Testing Laboratories,
local bakeries and supermarkets in Amman, Jordan.
|| Country of origin of analyzed maize and soybean food products
|GM indicates genetically modified products detected in this
Standard maize genetically non-modified (ERM-BF413a), genetically modified
MON 810 maize (BF413f), standard soybean genetically non-modified (BF410a) and
genetically modified Roundup ReadyTM soya (ERM-BF410e) were obtained from Dr.
Eric Kubler. These samples were originally purchased from European Commission,
DG JRC and IRMM, Belgium.
Genomic DNA extraction: Genomic DNA was extracted from flour, meal or
ground samples of maize and soybean products by Cetyltrimethylammonium bromide
(CTAB) method (Querci et al., 2006). Five hundred
microliter of CTAB was added to 100 mg of homogeneous sample already mixed with
300 μL of sterile deionized water. The mixture was subjected to protinase
K (20 mg mL-1) and RNase (10 mg mL-1) treatments at 65°C
for 30 and 10 min, respectively and then centrifuged for 10 min. The supernatant
mixed was with 500 μL chloroform and centrifuged for 15 min, this step
was repeated and the mixture was centrifuged for 5 min. Two volumes of CTAB
precipitation solution was added to supernatant, mixed gently by pipetting and
incubated at room temperature for 60 min then centrifuged for 5 min. The supernatant
was discarded and the precipitate was dissolved in 350 μL NaCl (1.2 M),
350 μL of chloroform was added and mixed for 30 sec followed by centrifugation
for 10 min. The upper layer was transferred to new microcentrifuge tube and
0.6 volume of isopropanol was added, mixed gently by inversion then centrifuged
for 10 min, the supernatant was discarded. Five hundred microliter of 70% ethanol
was added to the precipitate, followed by brief gentle mixing and centrifuged.
The supernatant was discarded and the DNA precipitate was allowed to dry, then
the DNA pellet was re-dissolved in 100 μL sterile deionized water. The
prepared DNA solution was divided into aliquots of 10 μL then stored at
-20°C for up to six months. Centrifugation was carried out at 16000 rpm
for aforementioned indicated periods.
||Sequences of primers used in PCR amplification experiments
(Querci et al., 2006)
Determination of concentration and purity of extracted DNA: The concentration
of extracted DNA was determined by measuring at 260 nm against a blank. The
ratio A260/A280 was used to estimate the purity of extracted DNA (Querci
et al., 2006). The measurements were performed using Jenway Spectrophotometer
Primers: The forward and reverse primers specific to zein gene (ZEIN3 and ZEIN4), CaMV 35S promoter primers: forward primer (p35S-cf3) and reverse primer (p35S-cr4) and primers for E35S promoter/hsp70 exon-intron cassette of maize MON810 (mg 1/mg 2, mg 3/mg 4); specific primers for soybean (GM03 and GM04) and soybean RRS (GMO5/GMO9, GMO7/GMO8) were used in the amplifications (Table 2). The primers were obtained from Alpha DNA/Canada.
DNA amplifications: Amplification reactions were performed according
to the reported methods (Querci et al., 2006).
PCR reactions were carried out in a total volume of 50 μL. Each reaction
mixture contained 5 μL of 10x PCR buffer, 5 μL of 25 mM MgCl2,
0.25 μL of Taq DNA polymerase which were obtained as TopTaq TM PCR kit
(Qiagen/Germany), 2.5 μL 4 mM dNTPs (Promega/Germany), 1.25 μL of
20 μM of each primers (Table 2), 32.75 μL nuclease-free
water and 2 μL of extracted DNA. The parameters of amplification were carried
out according to the reported protocols (Table 3). Each run
included standard genetically non modified maize and soybean, GM maize or soybean
products and no-template control containing all PCR mix component except DNA.
|| Parameters for PCR amplifications experiments for various
primers used for detection of GM maize and soybean food products
|ID: Initial denaturation, D: Denaturation, A: Annealing, E:
Extension, FE: Final extension, NC: No. of cycles, F: Forward primer, R:
Reverse primer, X indicates number of cycles during PCR amplification. The
amplification conditions were as reported by Querci et
The amplifications were performed in the Applied Biosystem Thermocycler 9902
with heating lid.
Gel electrophoresis: The amplification products in parallel with DNA
marker ladder of 100 bp (Qiagen) were separated on 1.5% agarose gel, run with
3 volt cm-1 and visualized under UV light after staining with ethidium
bromide for molecular size determinations in base pair (bp) of DNA fragments
(Sambrook and Russell, 2001).
Qualitative PCR-based methods were used to survey and detect the event of genetically
modified maize and soya products sold commercially in Amman, Jordan. The majority
of collected food products were maize products (Table 1).
Maize food products were mainly corn seeds, corn meal or flour, whereas soya
products were substitute for meat (soya meat), soy sauce and bread crumbs. Genomic
DNA was extracted by CTAB method from various maize and soybean food products,
the results showed noticeable variations in the concentrations and purities
of extracted genomic DNA. The study revealed four categories which were recognized
according to the yield of extracted DNA. The lowest yields range of extracted
DNA was 1 ng μL-1, whereas the highest yields range was
46 ng μL-1 (Table 4). The results of purity
of extracted DNA also showed variations and it varied between 1.49 and 1.93.
These results might suggest the suitability of CTAB method for DNA extraction
from maize and soybean products. Agarose gel analysis of extracted DNA from
various maize and soy products showed that the isolated DNA fragments by CTAB
method composed of high molecular weight and a smear of lower molecular weight
degraded DNA (Fig. 1a, b).
||Appearance of extracted DNA fragments on agarose gel following
exposure to UV light of 365 nm, the obtained concentration and purity of
extracted DNA by CTAB method from maize and soybean food products
||Agarose gel electrophoresis of extracted DNA from (a) maize
and (b) soybean food products by CTAB method. Electrophoresis was performed
on (1.5%) agarose gel and run with 3 volt cm-1. Lane's numbers
represent maize and soybean food products used in this study
||PCR amplification of zein sequence (277 bp) of maize food
products. Electrophoresis was performed on 1.5% agarose gel and run with
3 volt cm-1. Lanes from 1 to 12 represent the maize food products
||PCR amplification of lectin (118 bp) sequence in soybean food
products. Lane L indicates the 100 base pair ladder; Lanes 1-3 indicate
the soybean food products
Polymerase chain reaction experiments for the amplification of DNA sequences
by the specific primers (Table 2) showed that 4 out of 22
maize and soy products are genetically modified. The plant origin of the food
products was confirmed by using two genes specific for maize or soybean. Zein
gene was identified in maize food products (Fig. 2), the size
of amplified DNA fragment is 277 bp which is specific for zein gene. Whereas
lectin gene was identified in soybean food product (Fig. 3),
the size of amplified DNA fragment was 118 bp which is specific for lectin gene.
||Detection of PCR amplified 35 S sequence (123 bp) in (a) maize
and (b) soybean food products. Electrophoresis was performed on 1.5% agarose
gel and run with 3 volt cm-1. Lane L, indicates the 100 bp ladder,
Lane's numbers represent maize and soybean products
DNA fragment for the 35S promoter (123 bp) was identified in three of maize
food products (Fig. 4a, b) and one of soy
The results of nested PCR experiments showed that the three maize food products
contained the hsp70 exon1/intron1 region of maize MON810 (Fig.
5a). The molecular size of the amplified DNA fragment by primers mg1and
mg2 was equivalent to 401 bp, while the size of amplified DNA fragment by primers
mg 3 and mg 4 was 149 bp (Fig. 5b). Figure 6
shows the molecular schematic details of 35S promoter/hsp70 exon-intron cassette
of maize MON810. The Roundup Ready soybean gene cassette (Cp4 EPSPS) was not
detected in the tested soy food samples.
||Detection of amplified nested products in maize food products,
(a) 401 bp sequence identified by mg 1/mg 2 primers; (b) 149 bp sequence
identified by mg 3/mg 4 primers. Electrophoresis was performed on 1.5% agarose
gel and run with 3 volt cm-1. Lanes L and L1 indicate the 100
bp ladder, Lane L2 indicates 50 bp ladder
||Schematic details of 35S promoter/hsp70 exon-intron cassette
of maize MON810. 401 bp 149 bp represent the amplified DNA fragments by
primers mg 1/mg 2 and mg 3/mg 4, respectively
Present results showed that 18.2% of maize and soy food products sold in Jordanian
markets are genetically modified. These were identified by specific primers
and included both common GM detection primers for 35S and event specific. The
amplicons of the specific sequences detected are used to build the different
GMOs and regulate expression of transgenes, such as promoter 35S and the terminator
NOS (Forte et al., 2005; Vijayakumar
et al., 2009). Furthermore, nested PCR system was found useful in
detecting the transgenic event, this system of detection was developed for specific
detection of transgenic plants (Cankar et al., 2008;
Zhang et al., 2007; Zimmermann
et al., 1998).
From the time when first generation of GMOs seed were released for plantation
in 1995 and their products entered the food and feed markets of various countries
in 1996 (Paarlberg, 2006), there have been reports questioning
the safety of these organisms and their products when used as food or feed.
This brought about an active debate and controversy on possible risks that might
be caused by such crops and their products to health (Al-jebreen,
2010; Patel et al., 2005; Seralini
et al., 2007) and environment (Kawata et al.,
2009; Knispel and McLachlan, 2010; Sanvido
et al., 2007). As a result two main methods for the identification
of GM food and feed have been reported in the literatures; PCR and Enzyme-Linked
Immunosorbent Assay (ELISA). PCR is the most accepted technique used worldwide,
it showed consistent results when using specific primers for the detection of
the regulatory sequence or structural gene in the inserted gene fragment (James
et al., 2003; Marmiroli et al., 2008;
Matsuoka et al., 2000). The designed primers
must possess some specific characteristics and can be used for GM product screening
and product-specificity detection. Thus, it might be convenient to consider
these methods for the detection and identification of GMOs. This would represent
a new field of diagnostics in which a great deal of development has already
In this study, it was possible to detect genetically modified maize and soybean
unlabeled food products sold in the Jordanian market and the genetic event MON810
of GM maize was identified. These results are believed being the first survey
for GM products in Jordanian market and might draw the attention for implementation
of adequate labeling measures of food products containing materials derived
from GM crops. The obtained results suggest the necessity for well-organized
efficient monitoring process of GMOs and the need for labeling GM food products
in Jordan. The developing world in general and Jordan in particular often lack
coherent policies in regard to GM products, hence there is a need to strengthen
the indigenous capability in molecular biotechnologies in order to survey and
assess the bio-safety of GM food. This will be achieved with the support and
assistance of well established international GM laboratories (Al-Hmoud
et al., 2008).
Unlabeled genetically modified maize and soya food products were detected in the Jordanian market. Amplified 35S promoter DNA fragment was found in both GM maize and soya products. Genetic event MON 810 was detected in the GM maize food product, while Cp4 EPSPS event was not detected in GM soy food product. The study showed the need for further concerted efforts in the detection and handling of GM products in Jordan.
The Royal Scientific Society of Jordan appreciates highly the fellowship granted to M. Ibrahim by International Institute of Education. The research team is also thankful to Dr. Eric Kubler from University of Applied Sciences Northwestern Switzerland for his collaboration in supplying the GMO and standard samples of maize and soy.
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