Trends in Food Science & Technology 9 (1999) 380±388
Review
Detection of
genetically
modi®ed organisms
(GMOs) by PCR: a
brief review of
methodologies
available
E. Gachet,* G.G. Martin,*
F. Vigneau* and G. Meyery
*
Euro®ns Scienti®c S.A., Rue Pierre Adolphe Bobierre,
B.P.42301, F - 44323 Nantes Cedex 3, France
y
Hanse Analytik GmbH, Fahrenheitstraûe 1, D-28359
Bremen, Germany
The discovery of the molecules that bear the genetic
information of any living organisms among which DNA
(DeoxyriboNucleic Acid) plays a central role has been a
revolution for life sciences. Taking the advantage of the
universality of the genetic code, researchers have suc-
ceeded to associate DNA sequences coming from di€erent
organisms using molecular biology techniques and to inte-
grate foreign DNA within plants. These genetically-modi®ed
organisms (the so called GMOs) have the ability to synthe-
sise some additional proteins which confer new properties
on them. Improving the protection of agricultural crops is
one of the sought advantages by the gene transfer.
According to the European regulation, the new foodstu€s
made from genetically modi®ed soya or maize, must be
labelled. The control of the labelling of such foodstu€s is
based on the detection of the foreign DNA sequences born
by the genetically-modi®ed organisms. One of the analy-
tical methods used for enforcement of this regulation is the
Polymerase Chain Reaction (PCR) method. The principle of
0924-2244/99/$ - see front matter Copyright # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 92 4 - 2 24 4 ( 9 9 ) 0 0 00 2 - 3
the PCR method is to multiply speci®c sequences of DNA,
making them detectable. This highly sensitive method o€ers
the advantage of detecting DNA molecules which are more
thermostable than proteins. # 1999 Published by Elsevier
Science Ltd. All rights reserved.
As soon as human beings settled, they started to grow
plants and breed animals. Step by step, they arti®cially
created new species by selecting some types of plants
and animals (criteria were yield, weight, strength, etc.).
However, up to a few years ago, human beings were
unable to control directly the biological process of this
creation which is based on a random mix of genetic
information through sexual reproduction. Though
human beings have improved their techniques to control
sexual reproduction, they have not yet acted directly at
the core of the biological mechanism. They did not
modify the genetic information that living organisms
harbour.
Life sciences have undergone enormous progress
since the discovery that molecules bear genetic infor-
mation. These molecules are called nucleic acids, among
which DNA (deoxyribonucleic acid) plays a central role.
The knowledge of the structure and the biochemistry of
DNA, and mechanisms of DNA synthesis (polymeriza-
tion, ligation, cutting) has allowed biologists to act
directly on DNA sequences. Researchers have taken
advantage of this easy manipulation of DNA and of the
genetic code to associate DNA sequences coming from
di€erent organisms. They have succeeded in building
new DNA molecules by recombining di€erent DNA
sequences with molecular biology techniques (Fig. 1).
DNA is like a magnetic tapeÐit can be cut, moved and
reinserted to create new or di€erent information.
Thanks to molecular biology, researchers have suc-
ceeded in integrating foreign DNA within, for example,
plant genomic DNA, but also in mouse or rat DNA.
Genetically modi®ed organisms, abbreviated as GMOs,
are the fruit of this research.
Advantages of GMOs
The chronology of gene expression is as follows. The
gene is transcribed into its messenger ribonucleic acid
(or mRNA). Then, the mRNA is translated into a pro-
tein. Thus, one protein corresponds to a speci®c gene.
Introducing a foreign gene in a plant confers this on
plant the ability to synthesize a foreign protein.
 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388 381

Based on the idea that any genetic information from
any source can be expressed in any organism, genetic
engineering has, for example, looked at improving the
protection of agricultural crops. Other sought advan-
tages include shortening the delay to obtain a new vari-
ety, improving the yield and quality of crops, producing
high value-added molecules (like pharmaceuticals or
vitamins or biopolymers for industry), and improving
the nutritional quality of plants (Tables 1 and 2).
The ®rst genetically engineered fruit sold on the mar-
ket was the well-known FlavrSavr tomato, which was
designed to soften more slowly. A slower softening

Fig. 1. Di€erent steps for the creation of a GM corn containing an endotoxin gene.

Table 1. GMOs on sale in 1997

Crops Property Country Company
Corn Insect resistence USA, Canada, Japan, EU Ciba-Geigy, Monsanto, Mycogen, Sandoz,
Northrup King
Herbicide tolerance USA, EU, Switzerland de Kalb, AgrEvo, Plant Genetic Systems
Male sterility USA Plant Genetic Systems
Soy bean Herbicide tolerance USA, Canada, Japan, EU, AgrEvo, Monsanto
Argentina, Switzerland
Tomato Ripening slower USA, GB Agritope, Calgene, DNA Plant Technology,
Monsanto, Zeneca
Potato Insect resistance USA, Canada, Japan Monsanto
Chicory Male sterility and Europe Bejo Zaden BV
herbicide tolerance
Squash Virus resistance USA Asgrow, Upjohn
Melon Virus resistance USA
Papaya Virus resistance USA Cornell U.
Rape seed Male sterility EU Plant Genetic Systems
Herbicide tolerance Japan, Canada, USA AgrEvo, Monsanto, Plant Genetic Systems
High level of lauric acid Canada Calgene
Composition of oil USA Calgene
Cotton Insect resistance USA, Mexico, Australia, Japan Monsanto
Herbicide tolerance USA Calgene/RhoÃne-Poulenc, Du Pont, Monsanto
Tobacco Herbicide tolerance Europe SEITA
382 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388
Table 2. GMOs under development in laboratories or undergoing ®eld testing
Quality Property researched
Agronomical Herbicide tolerance
Virus or fungus resistance
Production of an antifreeze protein
Slower ripening
Nutritional Low amount of nitrates
Richer in starch
Lower level of saturated fats in oil
High level of amino acids
Pharmaceutical Not producing an allergen
Higher levels of a natural anti-cancer agent
Producing a vaccine against hepatitis B
Producing anti-cancer and antioxidant agents
Synthesizing haemoglobin
Industrial Coloured ®bres
Less ®bres
process means that the tomatoes can stay on the vine for
longer which makes them tastier. Furthermore, toma-
toes with elevated levels of an antioxidant, lycopene,
may protect against cancer. Currently, the most widely
inserted genes in GMOs confer resistance to worms,
insects or to a herbicide. One of the aims has been to
create a plant that resists any chemical protection used
by farmers; for example soy bean or corn that are tol-
erant to herbicides like Round Up1. Round Up1 is a
non-selective herbicide which acts by entering the plant
and inhibiting an enzyme necessary for building
aromatic amino acids. The lack of these amino acids
kills the plant. Herbicide-resistant corn varieties
designed to increase yields are already cultivated and a
corn that has its own inbuilt insecticide is already sown
and harvested in the United States.
In addition, a lot of GMOs are under development.
For example, genetically-modi®ed corn altered to make
a healthier cooking oil by reducing its saturated fat
content is on the way. In the case of strawberries, add-
ing an antifreeze protein from the winter ¯ounder ®sh to
help them grow in cold climates is under development,
as are nutritionally enhanced strawberries with
increased levels of a natural anti-cancer agent, ellagic
acid. In the same way, for broccoli, a combination of
natural anti-cancer and antioxidant agents could make
this cruciferous vegetable essential eating as it would
prevent or slow down the aging of cells that form living
organisms. Soon, potatoes richer in starch will be used
to produce low-fat chips and crisps that one might ®nd
in the stores in 5 years' time. A higher starch level leads
to a lower fat content because the potatoes cannot
absorb as much fat when fried. Another fruit has been
an issue of scienti®c research: the banana. Scientists are
investigating whether genetically-modi®ed bananas
could produce a vaccine against hepatitis B. Bananas
New GMOs
Wheat, sun¯ower, beetroot, lettuce,
tomato, potato
Sun¯ower, beetroot, tomato, rice, melon,
squash, cucumber, tobacco
Strawberry
Banana
Spinach, lettuce
Potato
Corn
Soy bean
Rice
Strawberry
Banana
Broccoli
Tobacco
Cotton
Poplar tree
could also be made tastier if they can be induced to
ripen more slowly. Two other plants are being engi-
neered: the ®rst is a type of rice that would no longer
produce an allergen factor, and the second is a lettuce
that would have a lower amount of nitrate. Among
GMOs, there are also a rape seed resistant to fungi or to
herbicides, and GM chicory, papaya and squash.
The genetically-modi®ed plants also concern non-
food crops like cotton with coloured ®bres or tobacco.
Even the poplar tree is going to be genetically engi-
neered to improve raw material (cellulose) for making
paper.
Development of the market for GMOs
At the present time, genetically-engineered plants
have been approved by several countries: the US,
Canada, The European Union (EU), Switzerland, Aus-
tralia, Argentina, Brazil and Japan. In the US, 20
genetically engineered products were approved in 1997
and 25 others have been submitted for approval. It is
expected that in the next decade, 48 agricultural crops
will be genetically modi®ed. In the EU, 12 varieties of
GMOs have been approved to be sold on the European
market. Nine other GMOs are going to be approved
soon.
Currently, the two most cultivated GMOs are corn
and soya. In North America, genetically-modi®ed crops
represented a small area of farmland in 1996, as only 1.5
million ha were covered with GMOs. As early as 1997,
the cultivated area of genetically-modi®ed crops rose to
12.5 million ha (Fig. 2). This year, one-third of soy
beans planted in North America were genetically-mod-
i®ed, representing a two-fold increase over the previous
year. There is a similar trend for corn. A signi®cant
fraction of US corn, the staple constituent of biscuits,
cakes and cookies, is already modi®ed. A total of 8.3
 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388
Fig. 2. The increase in the cultivated area of GMO crops in North America.
million ha of genetically modi®ed corn (20% of the total
cultivated area) were planted in the US this year, up by
10% over 1997. It is forecast that the cultivated area of
genetically-engineered crops will be multiplied more
than three-fold by the year 2000, and that more than 66
million ha will contain transgenic plants by 2005 in
North America. Some estimates suggest that in 10 years'
more than 75% of major crops are going to be geneti-
cally engineered. Soy bean is a strategic crop for the
food industry as well as for international trade. Indeed,
soy beans are used today in 60% of all processed foodsÐ
from margarine to chocolate, ice-cream, convenience
and baby foods. In addition, North America produces
the majority of the world's soy beans, sending up to
40% of the crop to Europe, an estimated 30% of which
is genetically modi®ed.
Laws controlling the marketing of GMOs in the
USA and the EU
By now, many countries have introduced legislation
regulating the approval and release of GMOs. In the
US, three independent authorities are involved in the
regulation of the release of genetically engineered plants
and their use as foodstu€s: APHIS (Animal and Plant
Health Inspection Service), FDA (Food and Drug
Administration) and EPA (Environmental Protection
Agency). The APHIS controls movement between
states, importation and culture assays of GMOs that
might induce diseases in plants or be, by themselves, a
disease. The FDA makes rules for additives and new
foodstu€s, except meat, and products coming from
poultry farming. The FDA also makes rules for animal
medicines. The FDA is also concerned with the labelling
of food. Currently, this administration considers that
the composition of a food consisting of or derived from
genetically modi®ed plants does not di€er signi®cantly
from its conventional counterpart. Thus, the labelling of
the genetically modi®ed organisms is not mandatory in
the US.
383
By comparison the novel food regulations in the EU
will require the labelling of GMO products if they can
be distinguished from respective conventional products
by scienti®c and analytical methods [1]. So, since Sep-
tember 1998, labelling has been made mandatory for
foodstu€s containing ingredients derived from geneti-
cally modi®ed corn (the so-called `Bt-Maize' from
Novartis) or soy bean (the so-called `RR-Soy' from
Monsanto). As the genetically-modi®ed organisms bear
supplementary genetic information in comparison with
the conventional plants, the purpose of the analytical
methods used for enforcement is to target these foreign
DNA sequences. The control and detection of such
foodstu€s will be possible thanks to the Polymerase
Chain Reaction (PCR) method in addition to the con-
ventional protein detection tests using antibodies like
the ELISA-test (Enzyme-linked immunosorbent assay).
Detection of GMOs by the PCR method
Principle of the PCR method as applied to GMO
testing
The principle of this method is to amplify speci®c
sequences of DNA thanks to a pair of short DNA
sequences that ¯ank the region to be ampli®ed (these
are known as primers). The PCR method is based on
the molecular structure of DNA. The two strands that
form the DNA molecule have a helical structure. Both
of them are linked by periodic hydrogen bonds between
the nucleotides (the elementary blocks of the sequence).
Genetic information is like an alphabet based on four
letters (the nucleotides, also named bases, are A, T, C,
G). Each nucleotide binds to only one of three other
nucleotides (A to T and C to G, and conversely T to A
and G to C). The two strands are thus linked: a strand
binds to its complementary strand.
However, both strands of the double-stranded DNA
can be separated from each other, under the action of a
speci®c enzyme by breaking the links inside the region
where the new strand of DNA will be polymerized. This
384 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388
is the ®rst step of DNA replication in vivo. When DNA
has been separated into individual strands, single-stran-
ded DNA is cut by a speci®c enzyme. Then, the poly-
merization of DNA can start. DNA polymerization
may be divided into two steps. At ®rst, an enzyme called
DNA polymerase binds to the end of the cut strand.
Then, DNA polymerase synthesizes the new com-
plementary strand. Each strand serves as a template for
synthesizing the complementary strand. There is a
polarity in extension of the new strand. As a matter of
fact, the DNA polymerase links a new nucleotide to the
30 end of the extending strand, each strand having one
end called 50 and the other end called 30 . Finally, two
identical molecules of DNA are synthesized from one
DNA molecule. The two daughter molecules are also
identical to the mother molecule.
The PCR mimics those three natural steps (Fig. 3). (1)
The single-stranded DNA molecules are obtained by
heating the DNA solution to a temperature of 94 C
(enough to break the hydrogen bonds between the
strands): this is the denaturing step. (2) By decreasing
the temperature to around 55 C, a single-stranded
DNA fragment can match its complementary single
strand. Two short and arti®cially-synthesized DNA
sequences (around 20±30 nucleotides), named primers,
that ¯ank the region of interest to be ampli®ed bind to
or hybridize to opposite strands. This is the annealing
step. Template DNA, primers, DNA polymerase and
bases are submitted to the reaction at the beginning of
the whole PCR. (3) Finally, during the polymerization
Fig. 3. Scheme of the Polymerase Chain Reaction (PCR).
step or extension step, the DNA polymerase synthesizes
the new strand. The polymerization step that is carried
out at around 72 C succeeds thanks to a DNA poly-
merase functional at high temperatures. Early on, this
thermo-stable enzyme was extracted from the bacteria
strain Thermus aquaticus, which lives in hot water
springs. The standard PCR consists of about 20±50
cycles of denaturation (single-stranded DNA form),
primer annealing and DNA synthesis. The target DNA
sequence can be ampli®ed many times with these several
rounds of denaturating and polymerization of DNA.
Such a repetitive series of cycle results in the exponential
accumulation of a speci®c fragment whose termini are
de®ned by the 50 ends of the primers. Because the primer
extension products synthesized in one cycle can serve as
templates in the next, the number of target DNA copies
approximately doubles at every cycle. Thus, 20 cycles of
PCR yields about a million-fold ampli®cation. This
highly sensitive method allows rapid in-vitro ampli®ca-
tion of small amounts of DNA fragments. Under ideal
conditions, less than 10 copies of a speci®c DNA
sequence are sucient to be ampli®ed by PCR into a
readable signal. This amount is then enough for the
DNA to be analysed.
The kinetics of PCR depends on the concentration of
the DNA and on its quality (Fig. 4). The more con-
centrated the DNA, the more quickly the targeted
sequence is ampli®ed. Conversely, the more DNA is
fragmented, the less targeted sequence is ampli®ed
quickly because the starting of the exponential ampli®-
 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388 385

cation is delayed. However, this problem of DNA frag-
mentation has an e€ect only when highly processed
food is analysed. Frequently, the degree of fragmenta-
tion is checked before starting the PCR.
After the PCR, it must be determined if a fragment
with the expected length has been ampli®ed. The PCR
products are subjected to the standard electrophoresis
technique. If there is any doubt about fragment identity,
the ampli®ed fragment can be checked more precisely to
determine that the PCR product is de®nitely the
sequence of interest. Several methods may be used to do
this:
. a special gel electrophoresis technique which sepa-
rates the DNA fragments according to their length
as well as according to their base composition, e.g.
using bisbenzimide-PEG as a ligand of a speci®c
DNA sequence [2]
. the DNA ampli®ed may be digested by an enzyme
that cuts the expected fragment into subfragments
whose size is known
. the Southern blotting technique (the PCR pro-
ducts are tested with a probeÐthe target DNA
synthesized arti®cially)
. the fragment ampli®ed may be sequenced to check
the order of base pairs of the DNA sequence.
The principle of the mainly used standard electro-
phoresis technique is the following: once the DNA target
is ampli®ed, the DNA solution is loaded on an agarose gel.
As they are negatively charged in the running bu€er, DNA
molecules will move through the gel under the in¯uence

Fig. 4. Kinetics of PCR.

Fig. 5. Example of standard electrophoresis of DNA: demonstration of the presence as well as the size of ampli®ed DNA molecules (amplicons).
386 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388

of an electric ®eld. The segregation of the di€erent frag-
ments of DNA is based on their size (Fig. 5). The smaller
ones are seen at the bottom of the gel. After the electro-
phoresis is done, DNA is stained using a ¯uorescent mole-
cule that binds to DNA and visualized under a UV light.
degrades proteins non-speci®cally as it cuts the
covalent bond that links amino acids)
. the crude DNA solution goes through a column
containing the resin
. DNA puri®ed is eluted from the resin.

Di€erent methods of DNA extraction used for GMO
analyses
Before the ampli®cation, DNA must be extracted
from samples to analyse. It is a puri®cation step in
which other types of molecules (proteins, lipids and
polysaccharides) are thrown away. A lot of protocols
allow this, but there are two main principles of extrac-
tion. Either, the crude solution of DNA is cleaned from
impurities by di€erent agents, or DNA molecules are
®xed on a resin with high anity for them before being
eluted. The CTAB-method [3] (CTAB means Cethyl-
trimethyl-ammonium-bromide) is based on the ®rst prin-
ciple, and the Wizard-extraction method [4] is based on
the second principle.
The CTAB-method is divided in ®ve steps:
. solubilization of DNA by addition of the CTAB-
bu€er made with the detergent CTAB (at a ®nal
concentration of 20 g/l)
. denaturation of a large amount of proteins con-
tained in the sample
. ®rst precipitation of DNA with the second CTAB
solution (the concentration of CTAB is lower: 5 g/l)
. second degradation of residual proteins
. second precipitation of puri®ed DNA with alcohol
addition.
The Wizard-extraction method is divided in three
steps:
. solubilization of DNA by addition of the extrac-
tion bu€er and proteinase K (an enzyme that
The CTAB-method is the basis for an ocial German
Method [5] and the Wizard-extraction method has
become the ocial Swiss Method [6].
Choice of di€erent target DNA sequences
Taking account of genetic building of GMOs, several
speci®c sequences can be detected. These sequences can
be classi®ed in three categories.
. First category: sequences that regulate expression
of transgenes. They are often formed by the
sequence of the promoter P35S (a promoter
extracted from the cauli¯ower mosaic virus which
regulates the transcription of this virus) and by the
sequence of the terminator Tnos (Tnos that
regulates the end of the transcription of the nopa-
line synthase, comes from the bacteria strain
Agrobacterium tumefaciens). Both of them are
widely used to build the di€erent GMOs.
. Second category: the genes that are used as genetic
markers to be followed the transgene through the
whole process of genetic building. They may, for
example, confer antibiotic resistance to GMOs.
. Third category: the target genes (transgenes) the
most widely introduced in plants are genes con-
ferring herbicide tolerance to plants (e.g. the gene
that codes a phosphinothricine acetyltransferase)
and a gene conferring an insecticide tolerance to
plants (the gene codes a -endotoxin, cryIA(b)). In
the frame of the European market, the plants cur-
rently approved and sold contain these genes.

Table 3. Some primers used for DNA ampli®cation

Test Set of primers used Target DNA sequence Size of the PCR product
Screening 35S-1/35S-2 35S promoter 195bp
NOS-1/NOS-3 NOS terminator 180bp
``Roundup Ready'' soybean RR01/RR02 EPSPS gene 508bp
RR04/RR05 EPSPS gene 179bp
GMO5/GMO9 EPSPS gene 447bp
GMO7/GMO8 EPSPS gene 169bp
p35s-f2/petu-r1 EPSPS gene 171bp
Soya GM01/GM02 Lectin gene 413bp
GM03/GM04 Lectin gene 117bp
``Maximizer'' maize CRYIA1/CRYIA2 Endotoxine gene 420bp
CRYIA3/CRYIA4 Endotoxine gene 189bp
Cry03/Cry04 Endotoxine gene 211bp
Maize ZEIN1/ZEIN2 Azain gene 485bp
ZEIN3/ZEIN4 Azain gene 277bp
ivrI-F/ivrI-R Invertase gene 226bp
 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388
Examples of primers
The detection of P35S or Tnos sequences may be
carried out with two sets of standard primers. The pri-
mers 35S-1 and 35S-2 are the forward and reverse pri-
mers for an ampli®cation of a 195bp-fragment of P35S
(Table 3). NOS-1 and NOS-3 are the forward and
reverse primers for an ampli®cation of a 180bp-frag-
ment of Tnos (Table 3). The detection of both sequences
forms the screening method [7]. The detection of the
gene enabling neomycin (an antibiotic) resistance may
be carried out with the primers Tn5-1 and Tn5-2 [8]
(Table 3). The detection of a speci®c gene that confers a
herbicide resistance (the gene encodes the enzyme 3-
Enolpyruvyl-Shikimate-5-Phosphate-Synthase [EPSPS],
i.e. a phosphinothricine acetyltransferase) may be car-
ried out with the pair of primers RR01 and RR02 [4], or
with GM05 and GM09 [9] (Table 3). These primers give
rise, respectively, to a 508bp-amplicon and a 447bp-
amplicon. P35s-f2 and petu-r1 [10] amplify a 171bp-
fragment which contains the arti®cial link between the
35S promoter and a part of a gene from petunia
(Table 3). The fragment is typical for Monsanto's
Roundup Ready resistant soy. The detection of the gene
that codes cryIA(b) may be carried out with the pair of
primers CRYIA1 and CRYA2 [11] or Cry03 and Cry04
[12] (Table 3). These primers give rise, respectively, to a
420bp-amplicon and to a 211bp-amplicon.
Methodology for the analysis of a sample
To monitor the process of detecting DNA and the
ampli®cation of sequences, several controls are neces-
sary. There are positive controls using primers which
will amplify a fragment that is in any case contained in
the plant under investigation. They are designed for
checking the quality of DNA preparation and the
appropriateness of the general chemical parameters for
DNA ampli®cation. The tests must give a signal after
the ampli®cation and staining steps. A failure in the
detection means that either there are inhibiting factors
or there is not enough DNA in the DNA solution or the
parameters used for the PCR are not right. For the
analysis of a foodstu€ made from corn, the primers may
be ZEIN1 and ZEIN2 [11] or ivrI-F and ivrI-R [12]
(Table 3). The aim of these two sets is to detect, respec-
tively, the gene coding azain or the gene coding inver-
tase (corn-speci®c genes), in transgenic as well as in
conventional corn. The control for foodstu€ derived
from soy bean may be the pair of primers GM01 and
GM02 [13] (Table 3). This set ampli®es a 413bp-frag-
ment of a soy-speci®c gene that codes lectin. These
primers give rise to a 413bp-amplicon. This product
is detectable in conventional soya and in transgenic soya.
Another positive control must be carried out to check
that the parameters of the PCR (choice of primers,
annealing temperature, time for each step of PCR,
number of cycles for DNA ampli®cation) chosen for the
387
speci®c reaction are suitable. A plasmid bearing a spe-
ci®c sequence of GMOs (P35S, Tnos or an other speci®c
sequence of the genetic modi®cation) may be used as a
control DNA. In order to perform this test, the DNA
sample can be divided in two halves. The control DNA
is then added to one of the two halves. PCR products
are expected at least with the trial containing the control
DNA. If no PCR products are generated at the end of
the PCR, it means that the PCR parameters chosen are
not right for the speci®c reaction or that inhibiting fac-
tors might still be present.
The negative control is designed to check that there is
no contamination by modi®ed DNA in the laboratory.
This trial does not contain any DNA. If a signal is pro-
duced it means that the procedures preventing con-
tamination of the materials and solutions used for DNA
extraction and ampli®cation have failed.
It is also necessary to be careful about false positive
or negative results. The detection of the P35S sequence
alone is not proof that the foodstu€ is made from a
GMO. As a matter of fact, a conventional plant con-
taminated by the cauli¯ower mosaic virus (P35S is ori-
ginated from this virus) will also give a signal with the
primers 35S-1 and 35S-2. Another explanation that may
be given is that the primers can hybridize to non-speci®c
sequences. In this case, the amplicon yielded does not
correspond to the fragment of the targeted sequence. To
prevent the misinterpretation of the results, sets of pri-
mers targeting other speci®c sequences from the geneti-
cally engineered organism must be used. DNA may not
be visualized with a ®rst set of primers if DNA is highly
damaged. This problem may be resolved by applying
the nested-PCR technique. When the ®rst round of
DNA ampli®cation has failed to produce a visible
amount of amplicon because DNA is too fragmented, a
second PCR is carried out using the outcome of the ®rst
PCR as starting material with two other primers that
can match to the DNA sequence within the region tar-
geted the ®rst time. In the case of a successful assay, a
smaller fragment will be produced. The following sup-
plementary primers may be used (Table 3):
. GM03/GM04 [13] (conventional and genetically
modi®ed soy bean) yields a 118bp-amplicon
. RR04/RR05 [4] or GM07/GM08 [9] (genetically
modi®ed soy bean) yields a 179bp-amplicon or a
169bp-amplicon
. ZEIN3/ZEIN4 [11] (conventional and genetically
modi®ed corn) yields a 277bp-amplicon
. CRYIA3/CRYIA4 [11] (genetically modi®ed corn)
yields a 189bp-amplicon.
What is the bene®t of detecting DNA instead of
proteins?
Nucleic acids are not considered as an interesting
class of compounds in food chemistry. Indeed, the
388 E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388
nucleic acids present in food have no nutritional value
and there is no direct relationship between DNA and
food quality. But whereas proteins are thermo-sensitive
molecules, nucleic acids are very thermo-stable mole-
cules. Upon processing, food proteins are no longer
detectable or detectable with diculty because they are
degraded. Conversely, nucleic acids are only slightly
damaged by heat treatment. In addition, the PCR
method is more sensitive than conventional protein
detection tests. By comparison, the ELISA-test, based
on using antibodies against speci®c proteins, a techni-
que widely used in pharmaceutical tests, may be around
100 times less sensitive than the PCR method [13]. Fur-
thermore, antibody tests such as ELISA are much more
laborious and time consuming to develop and to vali-
date than nucleic acid tests. So, thanks to this powerful
and precise tool, a speci®c nucleic acid can be detected
whatever the foodstu€ analysed, even in mixtures where
the GMO ingredient is present in low concentrations.
Another argument may justify the choice of DNA
rather than protein detection. That is, whereas certain
proteins may be expressed only in speci®c parts of the
plant, like leaves, beans, pollen or stems, the whole
genetic information is present everywhere in the plant,
because the same genes are present in each cell of the
plant.
Thus, a broad range of products can be analysed by
the PCR technique. Among the foodstu€s made from
corn, there are products like corn germ, corn ¯our, corn
pasta product, starch ¯our, whole corn, corn ¯akes,
popcorn, corn chips, cakes, cookies, baked products
and, if not in any case, even sugars derived from corn
starch which can be analysed for a genetic modi®cation.
The PCR technique is as well applied to a lot of matri-
ces made from soya like for example soy bean, soy
cream (liquid or lyophilized), soy milk (liquid or lyo-
philized), soy meal, tofu, meat products, soy ¯akes,
lecithin, soy protein concentrate and, in several cases,
depending on the degree and completeness of its pro-
cessing, even oil.
Conclusion
The genetic code is universal. It means that genetic
information is always borne by DNA whatever the
organism (bacteria, fungus, plant or animal). Thus,
DNA is easily recombined and transferred from one
organism to another. Furthermore, DNA is a
ubiquitous molecule as all the cells that form an organ-
ism contain the same DNA. In addition, DNA is a
resistant molecule, in the sense that it is quite resistant
to heat and acidity variations. All these properties
represent advantages for the detection of this mole-
cule in foodstu€s. Finally, very low amounts of DNA
may be ampli®ed thanks to PCR techniques, allowing
easy identi®cation.
Thus the PCR technique can target genes introduced
into the genetically engineered plants thanks to a set of
primers that amplify sequences from these cloned genes
or from regulatory sequences linked to them. DNA
fragments ampli®ed by PCR and having the expected
size, are the signature of the sample which is being
analysed. They indicate if foodstu€s are made from
genetically-modi®ed or conventional plants. This
enables us to determine, in case of positive result, with
which genetically engineered species the food product is
made.
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