detection of genetically modified...
TRANSCRIPT
Detection of Genetically Modified GrainsRD Shillito, Bayer CropScience, Durham, NC, USA
ã 2016 Elsevier Ltd. All rights reserved.
Topic Highlights
• The only major grains that presently contain genetically mod-
ified (GM) traits are maize (called corn in North America),
soybean, cotton, and canola (known as oilseed rape in
Europe).
• Since their introduction in the mid-1990s, GM grains have
been regulated in inconsistent ways in various trading
blocks and countries; different levels of GM content may
require labeling in different jurisdictions.
• Grain is traded as a fungible product by contract between a
buyer and a seller.
• Testing will normally only occur when the buyer requires
grain that is not stated to be GM but that may potentially
have a low content of GM grains.
• Testing can occur at many points in the supply chain. Tests
can be divided into two types, based on the target molecule:
– DNA-based methods such as polymerase chain reaction
(PCR) – based on the structure of the DNA
– Protein-based methods – based on the protein expressed
in the grain
• Production of grain with low GM content in a country with
a high-percentage GM crop requires a dedicated supply
chain.
• Obtaining a representative sample of the grain is a key step
in testing.
• Obtaining and preparing the grain sample are resource-
intensive and not amenable to improvement.
• Use of the same validated method by seller and buyer
reduces the chances of trade disruption, as does the use of
competent laboratories.
• The technology for performing the test for GM grain has
been evolving and continues to evolve.
Learning Objective
To achieve understanding of the approaches used to detect
genetically modified grains and the future directions of detec-
tion methods.
Introduction
A genetically modified (GM) grain is presently understood to
be a product developed through modern biotechnology by
insertion of a specific gene sequence into the plant DNA.
Such products have been described in the literature and by
governments and regulatory agencies in various ways – as
GM, genetically engineered (GE), GM organism (GMO), trans-
genic, biotechnology-derived, and recombinant. For the pur-
pose of this article, the term ‘GM’ grain will be used for its
simplicity and broad recognition.
Since their introduction in the mid-1990s, GM grains have
been regulated in differing ways in various trading blocks and
countries. Many jurisdictions require labeling of foods that
contain a level of GM materials above a nominated threshold.
These regulatory environments and the thresholds imposed are
not consistent across the globe and continue to evolve. Label-
ing for the presence of GM events (and stacks) in food is
required in many jurisdictions. Food producers and retailers
may desire not to have to label their product as containing GM
grain. This leads to the testing of grains to ensure that the GM
content of food (and in some cases feed) subsequently derived
from is below the threshold that would require labeling. Test-
ing is performed by screening for macromolecules indicating
the presence of genetic modification. It is carried out by traders
and their customers for the grain and also by regulatory author-
ities that are monitoring incoming shipments for unapproved
GM events.
Testing may occur at several levels: the seed that a farmer
plants, the grain delivered to the elevator, the grain that passes
through the supply chain, and the grain with only a low level of
genetic modification required by a customer. It may be tested
before export, at receipt in the market buying the grain, and
even at subsequent distribution points.
The level of GM content that may require labeling differs
between jurisdictions: 0.9% (based on weight by weight but
measured by DNA content) in the European Union, 5% (based
on % of the grains) in Japan, and somewhere between these
two such as 3%. This labeling is not on the basis of safety, as
the grain is approved in these jurisdictions for consumption at
100%; it is merely a food labeling requirement.
An additional state of affairs occurs for GM events that have
not yet been approved for consumption in a particular country
and are therefore not allowed to be present in grain sent to this
country. Any presence of the specific nonapproved event in a
grain shipment is not acceptable, and the shipment will be
rejected. This may occur when an event is approved for culti-
vation and grown in an exporting country, but not yet
approved in a country that imports grain from that country.
One example of this was MIR162, an event that was not
approved for import into China until late 2014 but had been
commercially grown in the United States for some years.
The major grains that are presently grown as GM grains are
maize (called corn in North America), soybean, cotton, and
canola (and oilseed rape in other areas). So far, crops of GM
wheat and other grains have not been commercially grown,
although all have been transformed in the laboratory, and
many have undergone research field trials.
This article will cover techniques used for detection of GM
grains in the major crops in which the technology has been
applied, though the principles are applicable to other crops as
well. The examples given are applicable for the detection of GM
grains in canola, soybeans, and maize (as these are the major
GM crops that are commercially traded internationally in large
Encyclopedia of Food Grains http://dx.doi.org/10.1016/B978-0-12-394437-5.00219-9 1
quantities. Cotton grain is not extensively traded on the inter-
national market as it has a low value compared to the cost of
transport, but the same principles of testing can be applied.
As a rule of thumb, a majority of grain is not tested for the
presence of GM grain, as a large amount is used as commodity
grain in the country of production. In addition, much of the
soy and maize grain that is exported from these countries is
exported for use in animal feed and may not be extensively
tested, except in the case where it is suspected, an unapproved
event may be present. Thus, testing of lots for the presence of
GM grain is an exception, rather than the rule.
In addition, testing occurs for purposes of making sure that
seed lots contain the transgenic material to the level that is
specified on the label, such as >98% purity (what is called
positive testing). This is not the topic or within the scope of this
article.
The Role of Testing
Traders have been testing grain for as long as grain has been a
food. When one person or organization (farmer or grain mer-
chant) is selling a grain to a second, they will agree on a price
based on a number of factors, including the grain quality. In
certain grains such as wheat, a key factor may be the expected
milling quality of the grain and/or protein content. The tests
performed in order to confirm that grain meets the buyer’s
expectations range from relatively simple tests such as for
odor (e.g., a smell test is part of the battery of tests performed
on all maize samples leaving the United States) to visual tests
such as examination for foreign seeds or stones; relatively quick
equipment-based tests for moisture, protein, and oil quality;
and tests using complicated machinery and sophisticated lab-
oratories. The buyer may specify the maximum (or minimum
in the case of value-added products) level of a particular GM
event in the grain to be delivered.
As stated previously, testing can occur at many points in the
supply chain. The tests performed will be influenced by the
situation. For example, tests performed at the delivery of grain
to an elevator by the farmer and at the point of export must be
capable of being performed in minutes, as the bulk grain is
awaiting transfer either out of a truck or into a ship or another
vessel. If a test taking more than a fewminutes is required, then
the sample for the test must be taken long before the result is
needed, so that the analysis can be performed while the grain is
in transit. Once a ship is loaded, it is not feasible (or economic)
to unload it in order to remove materials that have already
been loaded that do not meet the delivery contract. Thus,
testing for detection of GM grains usually occurs in the trans-
port chain well before the shipment is delivered to the cus-
tomer. The result of testing then confirms that the material
conforms to the intended use or, should GM grains be detected
at above the desired threshold, is diverted to a market that will
accept the material on the basis of that GM content.
Production of Low-Percentage GM Grain
The majority of maize, soybean, or canola in the major grain-
exporting countries is derived from GM grains. Almost all of
this grain is treated as fungible in that any source of grain can
be used to meet a contract.
Should the buyer require a low-percentage GM grain, they
will enter into a contract with the supplier as much as 2 years
before the delivery date. This long time frame is necessary as
the supplier will generally need to control the entire product
chain from the original seed grown to the final delivery to the
buyer and segregate the grain from the commodity material.
First, they must produce or obtain seed that has been certified
(and probably tested) to contain much less than the threshold
level of genetic modification desired by the buyer. This differ-
ence is to allow for any small increase in the presence of such
material due to adventitious contamination, which is almost
unavoidable in such identity-preserved product chains. The
supplier then must contract with a farmer to grow the seed
and produce the crop in a way that will limit the possibility of
ingress of seed or pollen that contains genetic modification
from neighboring farms.
The grain is then delivered to dedicated collection points
such as elevators using dedicated trucks and passes through a
segregated identity-preserved transport system, which may
consist of trucks, train cars, barges, and eventually oceangoing
vessels. In certain cases, the grain may be delivered in standard
shipping containers. This segregation of grain incurs significant
cost, from production of the seed to the dedicated specialty
supply chain that is needed to deliver it to the customer.
Contamination of the grain at any point by grain from the
commodity grain supply chain will reduce or eliminate its
added value as low GM grain.
Performing the Test
Obtaining a Sample
Both buyers and sellers will want to know that the grain
meets the specified contract. As with any analytic method,
obtaining a proper representative sample is a key step; an
analytic result is only as good as the sample on which it is
performed. It is clearly impractical to test every grain in a
lot, especially as the testing for GM grain is destructive, and
thus, a sample of the grain that is representative of the grain
lot must be examined.
There are various ways in which a sample can be obtained,
and these are well known and part of established grain sam-
pling practices. Experience has shown that the same sampling
procedures as used for other tests can be used to obtain a
sample suitable for determining the low-level concentration
of GM grain.
A truck-delivered grain will be sampled at various locations
in the load by probing the load, usually using a mechanical
probe system. Later, in the supply chain, the grain will be
sampled as it moves from one storage or transport location to
another. Sampling of moving grain is done using standard
procedures and apparatus that are also used to sample the
grain for grading the quality of the grain – that is, whether it
meets the grade and does not contain levels of toxins and
pesticides above acceptable levels. The same sample can be
split and used for these various purposes.
More detailed descriptions of how to obtain a representa-
tive sample are available. For further consideration of sampling
2 GENETICS OF GRAINS | Detection of Genetically Modified Grains
strategies, see Freese et al. and the USDA grain inspection
handbook.
The size of the sample that is processed to carry out the
test must be chosen so that the limit of detection is appro-
priate for the test used. If a test can only detect one grain in a
thousand, it would be inappropriate to grind 3000 grains
and test the resulting flour. The analyst would instead have
to test three 1000 grain test samples. Therefore, it is impor-
tant that both the sampler and the analyst understand the
limit of detection capabilities and limit of quantification of
the tests to be used.
Once the grain is processed to partial products such as flour
and oils, then sampling of the bulk materials can be done. The
issue at this point is that the limit of detection (lowest concen-
tration that can be detected) will depend only on the test – the
ability to define the number of grains that contributes to the
sample can no longer be controlled. Thus, if it is desired to
have materials that have low or undetectable levels of genetic
modification, then testing of the grain, rather than the final
processed food or feed material, is the most efficient and
effective approach.
The sample to be tested may be subsampled to provide the
laboratory sample that is to be tested. In order to perform the
test, the material will need to be ground to extract the protein
or DNA, in either water or in most cases a buffer. This must be
performed in a way that precludes contamination of the sam-
ple with other materials, to prevent false results. Sample prep-
aration for the test is not covered in this article, and
information is test-specific and can be reference to the test
procedure. Preparation of the sample for testing takes in most
cases more resources than the testing step itself.
Choice of Type of Test and Method
Two main analytic approaches are used to confirm that the
level of genetic modification in a grain lot does not exceed the
requested threshold. These are based on detection of the intro-
duced DNA or the protein(s) expressed in the GM grain.
Many jurisdictions insist on DNA-based analyses, specifi-
cally polymerase chain reaction (PCR), but some have used
protein-based tests to make sure that a lot meets regulatory
requirements. Testing a lot using protein-based methods can
offer significant cost savings.
DNA-based tests are almost exclusively performed in a con-
trolled laboratory environment. Thus, DNA-based detection of
genetic modification in grain is relatively expensive, and a
single sample can typically cost $200–400 USD to test at a
service laboratory, not including the cost of sampling the
grain and shipping the sample to the test facility. In addition,
a DNA-based (PCR) test typically takes at least 12 h and more
typically 2 days to complete. Testing by detection of the lateral
flow strip (LFS) protein test is relatively inexpensive (a few US
dollars per test), can be performed using simple equipment,
and takes less than half an hour.
As materials move though the product chain, their value
increases and the type of test that can and will be performed
changes (Figure 1). Raw materials can be tested using protein-
based tests. Such tests are not able to be used on materials that
have been processed or exposed to heat and/or moisture, and
the proteins may denature or degrade and no longer be recog-
nized by the antibodies that make up the test. As products
become more processed, then the preferred test changes to a
PCR approach.
Information about methods for DNA testing is available for
all of the commercially grown grain events from the producers
of the events themselves (CLI) and can be licensed formost uses.
This website also lists the protein-based tests that are available to
test for the presence of these products. Some countries/trading
blocks such as the European Union also publish methods infor-
mation that is for use only in meeting their regulatory
requirements and does not offer a license for other uses.
Qualitative Testing
A qualitative test produces a negative result if no target GM
seeds are in the sample tested and a positive result if one or
more of the target seeds are in the sample. An example of a
qualitative test is an LFS although some of these are now semi-
quantitative. In this approach, a subsampling strategy is often
used, in which a number of subsamples are tested. The number
of subsamples that give a negative result allows the level of GM
grain in the initial lot to be estimated. For example, if a contract
is for grain containing less than 0.5% genetic modification,
then a sample of 1200 grains may be split into three samples
and each one tested with a test that is capable of finding a
single GM grain in 400. Should two of three subsamples give a
negative result, then it is 95% certain that the lot contains less
than 0.5% of the grain with that characteristic. Such a test is
appropriate when some low level of the GM seed is acceptable
in a lot; the risks of rejecting good lots can be reduced by this
approach as it balances the risk to a buyer and supplier of the
grain (be it a farmer or a shipper). A test that does not tolerate
any positive pools imposes a high risk on the supplier side of
the contract and can lead to the rejection of good lots that meet
the threshold.
Where an event is not allowed in the grain at all (i.e., has
not been approved in the receiving country), then a test that
does not allow any positive result is appropriate. In this case, 3
Seed
Grain
p
p
p
Milled grain
d
d
d
d
dHalf product
Finished product
$$
$$$
$$$$
$
Supply ChainTesting
Figure 1 The value of material increases as it moves through thesupply chain ($ to $$$$). The type of tests applied also change(d, DNA-based test; p, protein-based test).
GENETICS OF GRAINS | Detection of Genetically Modified Grains 3
pools of 200 seeds would be necessary if the threshold was
0.5%, or the test can be performed on a single sample of 600
grains, should the test be sensitive enough to find that one seed
in 600. In general, thresholds for unapproved events are in the
0.1% range, so larger sample sizes (e.g., 3000 grains) are
required for 95% certainty and are more commonly carried
out using PCR.
Note that these determinations are statistical – it is not pos-
sible to completely guarantee that a lot does not contain a level
above the threshold or in the case of an unapproved event any of
that event. To do so would require testing every grain, which is
impracticable as the testing is destructive. Higher levels of cer-
tainty can be obtained using larger samples, which increases the
cost of sampling and testing. In addition, as the required thresh-
old decreases, then the sample size and/or the sensitivity of the
test required increases significantly. A threshold of 0.1% would
require the testing of 12 pools of 400 grains, with the lot being
accepted only if at least 11 of the pools gave a negative result.
Alternatively, a sample of 3000 seeds can be tested and returns a
negative result.
Qualitative testing is most often carried out using an LFS
and at country elevators, but qualitative PCR methods are also
employed to test for unapproved events in shipments further
along the transportation chain (barge/train).
Quantitative Testing
With quantitative testing, the acceptance limit can be any non-
negative value. The most common quantitative testing
approach is PCR, usually real-time quantitative PCR (RT-
qPCR). The mechanics of RT-qPCR are described in the side
pane [2].
As with qualitative testing, quantitative results also have a
degree of certainty. The uncertainty of a result (i.e., whether the
concentration of the lot is below the threshold) depends on the
uncertainty of the PCR result (which can be between 20% and
30%) and the uncertainty due to sampling of the lot. A typical
test would use 3000 seeds for determination of a concentration
in the 0.5–1% range, and quantitative results are not generally
possible nor reported by testing laboratories for samples with
less than 0.1% of GM grain.
LFSs have also been developed that give an estimate of the
quantity of an event in a lot. For some events, this can be quite
accurate, while for others, it is less so, as it depends on the
amount of expression of the protein that is detected in the
grain. The expression of the protein in grain from any particu-
lar event can vary from location to location, which can intro-
duce some uncertainty into the measurements.
Screening
When testing for the presence of GM events, especially by
PCR, a two-step process is often employed. The first step
involves screening for the detectable presence of GM crop
materials and the second, for the specific events that may be
present and to discriminate between those that are allowed
and not allowed.
Protein-based methods tend by default to be screening
methods. This is so because a particular protein (e.g., Cry1Ab)
may be expressed in more than one event. Only if a protein is
specific to one event can it be considered an event-specific test.
Screening is most often carried out by detection of DNA
elements that are common inmultiple events. In the early years
of GM crops, the 35S promoter and 3’nos elements were com-
monly used. The 35S sequence can be used to drive the expres-
sion of genes throughout the plant, and the 3’nos sequence
used to ensure correct termination of the DNA transcript. Thus,
screening of materials for the presence of these two common
elements allowed detection of most if not all events. In addi-
tion, the sequences of herbicide tolerance genes that are used in
multiple events can be used. With the advent of new promoter
and other elements, the usefulness of these screens is reduced,
although the 35S sequence is still commonly used to detect
nine or more events in maize. One caveat is that the 35S
sequence originates from a plant virus and is therefore com-
mon in certain species such as brassicas (including canola) that
can also be present adventitiously either in grain or in complex
foods such as soups. Therefore, caution in interpreting the
results of a test for the 35S sequence is particularly important.
Threshold Testing
Determining whether grain meets a threshold may be done by
using a quantitative test of either a single sample or a number
of subsamples. In all cases, these determinations have a degree
of uncertainty, and the normal approach is to make sure that
the lot has at least a 95% certainty of being below the required
threshold. In most cases, the threshold will be set below the
regulatory or final contract threshold to ensure that the actual
level always meets the regulatory limits. Each subsample can be
analyzed using a qualitative test or a quantitative test.
Analysis Based on the Protein Expressedin the GM Grain
Methods that detect proteins in GM grain are based on their
recognition by antibodies (Box 1). Just as for any other protein,
antibodies can be raised to specifically detect a particular protein,
for example, a Bt protein. So far, most genetic modifications that
are intended to confer herbicide tolerance or insect resistance to
plants involve expression of one ormore specific proteins. These
proteins are usually present in the grain in significant amounts
and can be used to detect the presence of such GM grain. Com-
mercial immunoassays are available for most of the GM grain
crops on the market today. Enzyme-linked immunosorbent
assay (ELISA) and LFS are themost commonly used test formats.
An LFS recognizes a specific protein in the grain, which is char-
acteristic of one or more GM events, but cannot differentiate
between multiple events that contain the same (or similar)
protein(s). LFSs are designed for relatively quick qualitative
yes/no testing. ELISA can be used as either a qualitative or a
quantitative assay.
The predominant use of protein-based tests in grain is as an
LFS. These commercial products are termed alternatively as
Lateral Flow Device, ImmunoStrip™, or QuickStix™ according
to the manufacturer’s preference and branding.
4 GENETICS OF GRAINS | Detection of Genetically Modified Grains
Analysis Based on the DNA in the GM Grain
Analysis based on DNA depends in most cases on using the
PCR technique (Box 2), although other DNA analysis methods
such as isothermal DNA amplification are becoming available,
PCR is able to amplify a small portion of the DNA present in
very low concentrations to make it visible, physically either via
staining with a fluorophore or via the generation of a fluores-
cent signal. PCR selectively amplifies specific DNA sequences
and can be used to detect and discriminate between traits and
between events. End-point PCR detection methods are used for
qualitative screening. RT-qPCR can be used for quantitative
detection.
As described by Lipp et al., PCR testing is used for specific
purposes in the grain handling/processing industry:
Box 1
Immunoassays employ antibodies as detecting reagents. Antibodies are
glycoproteins produced by specific cells of the immune systems of animals
in response to stimulation by a foreign substance. The foreign substance that
elicits the production of a specific antibody is referred to as an antigen. The
attribute of an antibody that makes it useful as a reagent in a diagnostic kit is
its capacity to bind specifically and with high affinity to the antigen that elicited
its production.
An ELISA consists of a first antibody that is absorbed to a surface of a
container (usually a multiwell dish (Figure 2)). The antibody is able to capturethe target protein from an extract of the grain. The rest of the proteins and other
components of the extract are washed off, and a solution containing the second
antibody is added. This second antibody also recognizes a protein. The second
antibody is attached to either a colored or fluorescent compound or an enzyme
that will produce such a compound, which provides a detectable signal or is
detected using a third antibody that provides the detection process (Figure 3).Other variations of this method also exist, but the goal is the same, to be
able to measure the presence and, when used quantitatively, the amount of the
target protein.
An LFS is essentially a solid-phase ELISA in which the target protein is
first recognized by the antibody linked to the detection system and then
captured by a second antibody at a specific position in the strip. One of the
most common consumer applications of this technology is a pregnancy test.
An extract of the grain is applied to the bottom of the strip in a vertical
position (Figure 4) and allowed to absorb into the sample pad. A positive
result is indicated by two lines and a negative by just the control line. If the
strip has been compromised, then the upper control line will not be present
and the test is considered invalid.
Figure 5(a) and 5(b) demonstrates how the LFS works: the target
protein (if present) is recognized by an antibody in the antibody conjugate
pad, which binds to it (Figure 5(a)). This first antibody is attached to a visualmarker, usually colloidal gold. The liquid is sucked up the strip and carries the
extract with it. As it passes over the position of the test line, the target protein
is captured by the antibodies. The target protein is also carrying the second
antibody that is attached to the colloidal gold. Thus, a line of colloidal gold
develops at the site of the capture antibody, only if the target protein is present
in the sample. The strip also contains a second line. This line captures the
remaining antibody (with the gold) and forms a control line. The control line is
present to show that the strip is performing correctly, as the antibodies are
proteins that are easily damaged by excessive heat, moisture, or solvents and
have a limited shelf life. Should the sample not contain the protein that is
being tested for, then the strip will only develop the control line
(Figure 5(b)).
Figure 2 A typical multiwell plate showing the result of an ELISA.The samples that contain target protein are indicated by the developmentof a yellow color from the action of the conjugated enzyme with achromogenic substrate.
Antibody toCry 1Ac
Cry 1AcAntibodyconjugate
Cry 1Ac
Enzyme toproduce color substrate
produces color
Figure 3 The ELISA test consists of a first antibody adsorbed on asurface, which recognizes the target protein (Cry1Ac in this case), and asecond antibody that recognizes the target protein. The color is typicallydeveloped by an enzyme conjugated to the second antibody.
PO
SIT
IVE
NE
GAT
IVE
INVA
LID
Figure 4 Typical lateral flow strips (LFSs) showing positive andnegative test results and a strip that has been compromised so that itgives no result.
GENETICS OF GRAINS | Detection of Genetically Modified Grains 5
Wicking Pad
AntibodyConjugate Pad
Positive Sample Labeled specific antibodies
NitrocelluloseMembrane
Antigen complexed toconjugated antibody
Target Antigen
Sample Pad
Gold-labeledAntibody
Backing MaterialControl
Line TestLine
Wicking Pad
Sandwich formation with capturing antibody
Unbound conjugated antibody with anti-conjugate antibody
ControlLine
TestLine
Negative Sample Labeled specific antibodies
Absence of antigen complexed to conjugated antibody
No Target Antigen
ControlLine
TestLine
ControlLine
TestLine
No sandwich formation with capturing antibody
Unbound conjugated antibody with anti-conjugate antibody
Figure 5 (a) Development of lines on an LFS in response to a positive sample. (b) Development of lines on an LFS in response to a negative sample.
6 GENETICS OF GRAINS | Detection of Genetically Modified Grains
PCR testing for unapproved events: An event may be approved for
use in the country of production, but not yet approved for
use in an importing country. In these instances, the import-
ing country often requires that the grain shipment be tested
for the presence of specific GM events to ensure that it does
not contain unapproved events. Such testing may rely on
qualitative PCR or a negative result in a RT-qPCR because in
most cases, there is no tolerance threshold.
PCR testing for GM content: Most countries that have adopted
mandatory labeling rules for food or feed have set labeling
tolerances for the presence of GMmaterial in grain products
or the final foods based on a percent GM (weight-to-weight
or % of DNA) content. In order to avoid labeling, the grain
used typically comes from an identity preservation program
and is certified to contain GM grains only at a level below
the desired threshold.
PCR testing for ‘non-GM’ labeling: In some countries (such as the
United States), food manufacturers and retailers wish to use
positive labeling for products that are described as not being
produced using GM products. In most cases, the use of
positive labeling requires that the grain and grain products
originate from an identity preservation program and test
negative or at least below a certain threshold for GM DNA.
Detection of the presence of GM grains based on the presence
of the DNA is often used to make sure that consignments
comply with contract conditions and for compliance to regu-
latory requirements. PCR is the specified regulatory method in
many but not all jurisdictions.
Validation of a Test and Proficiency Testing
For practical applications like grain testing, it is important that
the test performed gives the same result when performed in
different locations, for example, at export and import. This is
called transferability. The test must also be able to work when
there are small variations in reaction conditions, and use of
different PCR machines (robustness), as this is what will be
encountered in the real world. In addition, laboratories that
carry out routine testing will usually be operating under a
quality assurance system such as ISO 17025. Good laboratory
practices and quality standards such as ISO 17025 require that
a laboratory routinely test unknown samples and compare
them against the true value for those samples. The latter pro-
cess is called proficiency testing.
Validation
Validation of a test is done by performing the test at multiple
laboratories under the same conditions. For many methods used
for food analysis, this is carried out under the control of standardi-
zation organizations (SDOs) such as the AACCI, AOCS, and
AOAC, and the methods are published as ‘approved methods.’
This validation is carried out to standard ISO 5725, also known as
the ‘harmonized protocol.’ This can take up to 2 years to complete
and requires the participation of a large number (>12, typically
18–20) of laboratories. A large number of GM events are being
produced, for each of which an event-specific method is required.
The sheer number of these methods makes it impractical to carry
out validation via the standard protocols through SDOs. Hence,
validations are carried out internally by the developers, followed
by external validations on a limited scale. Then, national and
regional regulatory authorities such as the European Union typi-
cally validate these methods further. Laboratories and other orga-
nizations that have their own internal proprietary methods such
as screening methods validate them internally.
Proficiency Testing
Once a method is shown to work properly, it is put into use by
testing laboratories. Each laboratory that is testing grain in
trade should be able to show that it can perform testing in a
way that gives consistent results with other laboratories
Box 2
PCR technology was developed in 1983 by Kary Mullis. It is used to amplify
small pieces of DNA to generate thousands to billions of copies of specific
DNA sequences. Two main approaches predominate – gel-based PCR and RT-
qPCR. The identification and amplification mechanism is the same in both
cases, and RT-qPCR relies on the release of a fluorescent compound and its
measurement by a specific machine at every PCR cycle.
PCR can be used to detect genetic traits and discriminate between GM
events in grain.
Each GM trait or event is coded for by a specific DNA sequence inserted in
the plant genome. Selective amplification of the target DNA sequence is
accomplished using DNA primers that are specific to that sequence. A sample
of the DNA to be tested is added to a tube containing buffers, nucleotides, the
specific primers, and the enzyme that is capable of synthesizing copies of the
DNA (Taq enzyme). The mixture is subjected to repeated heating and cooling
cycles – 30–40� – and exponential amplification of a PCR product ensues
(side box). The PCR product for each set of primers and DNA target are of a
known size. In gel-based PCR, the DNA that is negatively charged can be
separated based on size using an agarose gel. Smaller fragments move faster
than larger fragments, thus giving a separation based on the size of the fragment.
The DNA is visualized using a compound that binds to the DNA (interchelator)
that fluoresces in UV light and can thus be used to visualize the bands. The
presence of a band of the expected size indicates that the target DNA is almost
certainly present. Use of appropriate positive and negative controls and, if
needed, sequencing of the fragment produced increase the accuracy of the
method. Thirty-five to forty cycles of heating and cooling are typically required to
generate a usable signal.
In real-time PCR, a third element is introduced. This is a short DNA
sequence primer to which are attached a fluorescent reporter molecule and a
quencher. In each PCR cycle, the reporter and quencher are released, and the
fluorescent reporter, as it is no longer quenched, is able to be detected and
measured. After each elongation cycle, the amount of this molecule is
measured, thus leading to a measure of the amount of the target DNA
sequence that is present in the sample. As in gel-based PCR, between 35
and 40 cycles are typically required to generate meaningful data. In order to
quantify the amount of a target DNA in a sample, the number of cycles required
to bring the signal above the instrument baseline is recorded. This cycle where
the signal crosses the threshold is termed the Ct value. A higher starting
concentration of the DNA target means fewer PCR cycles are required to cross
the threshold, and thus, the concentration of the target can be deduced by
comparison to a standard curve. RTPCR can also be used in an end-point
mode, where the signal is only read at the end of the reaction, and thus gives a
plus/minus result as provided by gel-based PCR.
There is a large amount of information on PCR available on the Internet,
including videos demonstrating how PCR works (e.g., https://www.youtube.
com/watch?v¼iQsu3Kz9NYo).
GENETICS OF GRAINS | Detection of Genetically Modified Grains 7
performing the same measurement. To do so, they participate
in proficiency programs. Organizers of proficiency programs
supply a set of materials containing amounts of the GM grain
that are the same for each participant, who at the time of
analysis does not know the concentration of the GM grain in
the sample(s). The participants return their results to the orga-
nizer who compares them against the intended values for the
material sent out and with all the other participants and then
provides the results of the comparison to each participant. In
this way, a participating laboratory can compare its perfor-
mance and measurements against those of the laboratory com-
munity and correct any bias in their measurements. The
proficiency testing process is common for other analytes in
grain. The two most important globally available GM grain
proficiency programs are those offered for maize and soy
grain by the US Department of Agriculture (http://www.
gipsa.usda.gov/fgis/proficiencyprogram.aspx) and for seed by
the International Seed Testing Organization (ISTA; www.seedt-
est.org).
Interpretation and Reporting of Results
Most testing results are clear and unambiguous – a GM grain is
found to be present or not present, and if an event-specific test
is applied, then the event is identified. However, it is important
to understand that in some cases results can be ambiguous,
and in all cases, care must be taken in interpreting results,
especially when the results have regulatory significance and
may result in economic consequences.
In protein-based testing, it must be considered that a par-
ticular protein, while present in grain, may be also present as a
consequence of the adventitious presence of grain of another
species. This is particularly important to consider in the case of
maize, soybean, and cotton, which have a significant number
of GM proteins that may be present in all three species. Protein-
based tests, such as an LFS, often rely on visual identification of
the result, whichmay be subjective. Testing should therefore be
performed by an experienced operator and in good light and
using the procedure specified by the manufacturer. The use of
readers to measure the density of bands on an LFS is becoming
more common and offers a more objective and repeatable
result when the strip is being used for testing large amounts
of grain. The issue of subjectivity may also arise when inter-
preting the results of PCR based on examination of a gel for the
presence of bands. Ambiguity can be reduced in both these
cases by limiting the size of each (sub)sample analyzed so as to
work well within the sensitivity of the method.
In the case of PCR testing, the most caution should be
observed when using the 35S sequence as a screening method.
As mentioned previously, this sequence originates from a plant
virus and is therefore common in certain species such as bras-
sicas that can be present adventitiously in grain. Here also, the
adventitious presence of other grains should also be consid-
ered – for example, a 35S promoter used in maize could be
found to be present in soybean samples that do not contain
any obvious GM maize, but that have traces of maize dust or
grains due to sequential use of the same trucks or shipping
containers in the soybean and maize transport systems.
PCR, because it is a very sensitive analytic technique, is prone
to cross contamination – from other samples, from the products
of PCR reactions, or from poor laboratory techniques. Thus,
many laboratories have adopted a forward flow process,
whereby the samples and the technicians follow a path through
the laboratory that is designed to prevent materials from each
step of the analysis contaminating a previous step. In the most
sophisticated laboratories, this involves the use of positive and
negative air pressures and pass-through hatches fromone area to
another to prevent passage of air during sample transfer. Thus, it
is recommended that specially designed facilities be used when
looking for low levels of GM grain, particularly when the results
are being used for regulatory purposes.
Concordance of Results from Different Kinds of Tests
If a sample is to be tested using several tests, particularly both
protein-based and PCR tests, then there should be rationale for
doing so. This may be that a protein test can screen for multiple
events, but a PCR test is required to detect specific materials.
Tests may also be done in sequence, such as screening, fol-
lowed by specifically analyzing for certain events (such as those
not allowed in the import country). It is expected that results of
such tests should agree, if carried out on the same material. It is
important to note that two grain samples from a lot are not
identical and may yield different results, so where possible, all
the tests should be carried out on one sample of the grain,
ground in such a way so as to make all the subsamples identi-
cal. With very low concentrations of GM grains, even this may
still lead to disagreement between analyses due to differences
in the sensitivity of tests, and thus, a thorough testing strategy
should be devised before starting the process.
In quantitative testing, the way that analyses are performed
will influence the results. Should the laboratory test on the basis
of subsampling, then the result will be in terms of the mass
fraction (percent) of grains. RT-qPCR is the method most often
used for regulatory purposes, and subsampling-type approaches
are most commonly used for receipt of grains at elevators in
identity-preserved grain programs. If RT-qPCR is used, then the
result will be in DNA copy numbers or percent of DNA repre-
sented by the genetic modification in the grain. It is not atypical
for such numbers to be different by a factor of two. For example,
one heterozygous grain in 100 (1% by grain count) will be
measured as 0.5% using RT-qPCR as it represents only 0.5% of
the DNA present. If the single grain were homozygous, then the
results would both be 1%. This difference must be understood
and accounted for when interpreting results.
Reporting of Results
A laboratory that is testing grain will report the results to the
customer. This report will include the type of test performed
and the result (yes/no/amount) and in the case of a quantita-
tive result should include the uncertainty of the result. This
uncertainty value reflects the fact that no measurement is
completely accurate but is an estimate of the actual amount
of GM grain present in the sample. The report will generally
not include information about what range of GM grains may
give rise to the result (as in a screening test) and will not, if
compliant to most international standards, include phrases
8 GENETICS OF GRAINS | Detection of Genetically Modified Grains
such as ‘GM-free’; the correct term for reporting a negative
result is ‘nn not detected,’ with ‘nn’ being the analyte (protein
or DNA sequence) that has been tested for.
Thus, the submitter of the sample also has the need to
understand and interpret the results and/or must work with
the analytic laboratory to understand its ramifications and
then add the uncertainty introduced by the sampling step.
Future Prospects
The technology for testing grain for the presence of GM grain
has been evolving and continues to do so. The methods in the
market today include LFS, ELISA, and both qualitative and
quantitative PCRs.
In LFS testing, we are seeing increased reliance on readers to
read the strips and gather the data, which makes measuring the
presence of genetic modification in grain more objective and
the results more traceable. In addition, these readers can yield
quantitative results for certain events. However, there are new
challenges; multiple events contain the same protein, often at
different concentrations, which can make interpretation of
results of protein tests, particularly semiquantitative results
from readers, complicated. In addition, LFSs are increasingly
being used in certain situations to test for the presence of
multiple proteins using one strip. For grain testing, it can be a
challenge to make this combination strip sensitive enough,
but manufacturers are continuing to develop the technology.
New events that do not express a protein that can easily be
detected are being commercialized, and new approaches based
on DNA are being developed for fast detection of these kinds of
events.
While RT-qPCR is the standard regulatory approach in most
wealthy economies, it can be difficult to obtain and maintain
quantitative PCR machines in less-developed economies. Pur-
chase of the supplies, training of the operators, and particularly
service of these machines can be a significant burden. Thus,
there are many places where gel-based PCR is the preferred
approach. In addition, gel-based methods have been routinely
used for screening in some of the most sophisticated testing
laboratories in the world, as they have found them to be robust
and cost-effective.
Thus, we have the situation where we are faced with multi-
ple protein and DNA-based methods used for testing in the
grain trade. We must appreciate that they have the potential to
give conflicting results.
Although there are a number of technologies being sup-
plied in clinical and research situations for performing multi-
ple and advanced protein analyses, these are not suitable and
are not being applied in the grain trade. However, two DNA-
based technologies, isothermal DNA amplification and digital
PCR, are maturing to the point where they may be deployed for
testing purposes.
Isothermal DNA amplification is a technology that allows
detection of specific DNA sequences (much as in PCR) in
minutes, rather than hours. There are at least five technologies
of which RPA (recombinase polymerase amplification), LAMP
(loop-mediated isothermal amplification), and NEAR (nicking
enzyme amplification reaction) appear to be the most
advanced in terms of application to plant systems. As of early
2015, no commercial kits using these technologies are avail-
able for GM testing in grain.
Digital PCR is another application of PCR that has the
potential of providing high-throughput analysis and the abil-
ity to analyze for multiple DNA sequences efficiently. Digital
PCR has a high potential and has already been deployed
for breeding purposes. As yet, it has not been used for routine
testing of samples for the presence of GM grain, and the
question will be whether it or conventional real-time PCR
will be more efficient in real-world applications, especially
in terms of the cost of purchasing and operating the
equipment.
It is important to understand that although the test
methods may develop, preparation of a sample that is typically
in the kg range for testing in the laboratory takes significantly
more resources, and little has and can be done to make this
step more efficient.
Conclusions
While sampling and testing of grain have been occurring since
the advent of grain trading, detection of GM grains in grain
shipments is an additional step that was added recently and is
taken if the customer desires them to be absent (or present in
the case of valuable consumer traits) at a desired concentration
above or below a certain threshold. The reason for these
thresholds may be preference, adherence to regulations, or a
combination of both. Those testing their grain lots have to take
into account the uncertainty introduced by (1) the fact that
they must rely on a representative sample of the grain to
estimate the concentration of GM grains and (2) the uncer-
tainty of the result produced by the methods themselves, be
they protein- or DNA-based.
The amount of testing required to meet local regulations
continues to increase as more crops are launched and more
countries grow these crops. The application of new method
approaches for detecting GM grain will continue to evolve, but
testing resources may be limited, and where they are limited
may be better applied to other more safety-related characteris-
tics of grain and food.
Exercises for Revision
• What are the grain crops that are GE in the market and
internationally traded?
• Are there internationally recognized ways of sampling
grain?
• Name four of the steps at which grain may be tested.
• Why must methods be validated? What is the role of profi-
ciency testing?
• What kind of test is suitable if you need an answer in half an
hour?
• What kinds of methods can be used for screening
shipments?
• What precious metal is used in an LFS?
• What is the result of the test if no lines develop on an LFS?
• How are ELISA and LFS tests similar and different?
• What is the meaning of Ct in relation to PCR?
GENETICS OF GRAINS | Detection of Genetically Modified Grains 9
• How many PCR cycles are generally needed to get a mean-
ingful answer?
• How are protein-based tests modified/adapted to make
them more quantitative?
Exercises for Readers to Explore the Topic Further
• How is digital PCR different or similar to other PCR
approaches?
• How does isothermal DNA detection differ from RT-qPCR?
• What methods might be used in the near future for testing
grain for the presence of GMOs?
• How is sampling of grain performed and what statistical
principles are involved?
See also: Food Grains and the Consumer: Genetically ModifiedGrains and the Consumer; Grain Composition and Analysis: TheStandardization of Methods for Analyzing Grains and Grain-BasedProducts ; Grain Harvest, Storage and Transport: Stored Grain,Handling from Farm to Storage Terminal; Genetics of Grains:Development of Genetically Modified Grains.
Further Reading
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Freese L, Chen J, and Shillito RD (2015) Sampling of grain and seed to estimate theadventitious presence of biotechnology-derived seeds in a lot. Cereal Foods World60(1): 9–15.
Grothaus GD, et al. (2006) Immunoassay as an analytical tool in agriculturalbiotechnology. Journal of AOAC International 89: 913–928, Available online athttp://aeicbiotech.org/wp-content/uploads/2014/08/AEICproteinpaper_2006.pdfISTA proficiency test on GMO testing: http://www.seedtest.org/en/proficiency-tests-content-1-1157.html.
Horwitz W (1995) Protocol for the design, conduct and interpretation of method-performance studies (Technical Report). Pure and Applied Chemistry 67(2): 331–343.
Laffont J-L, et al. (2005) Testing for adventitious presence of transgenic material inconventional seed or grain lots using quantitative laboratory methods: Statisticalprocedures and their implementation. Seed Science Research 15: 197–204. http://dx.doi.org/10.1079/SSR2005210.
Lipp M, et al. (2005) Polymerase chain reaction technology as an analytical tool inagricultural biotechnology. Journal of AOAC International 88: 136–155, Availableonline at http://aeicbiotech.org/wp-content/uploads/2014/08/polychnrctn.pdf.
Privalle L, et al. (2012) Development of an agricultural biotechnology crop product:Testing from discovery to commercialization. Journal of Agricultural and FoodChemistry 60(41): 10179–10187. http://dx.doi.org/10.1021/jf302706e.
Privalle L (2015) Development of genetically modified grains. Chapter 00218,Encyclopedia of Food Grains, Genetics, vol. 4.
Remund K, et al. (2001) Statistical considerations in seed purity testing for transgenictraits. Seed Science Research 11: 101–119. http://dx.doi.org/10.1079/SSR200166.
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Taylor JL (1987) Optimizing the expression of chimeric genes in plant cells. Molecularand General Genetics 210: 572–577.
USDA GIPSA Proficiency program: http://scl.gipsa.usda.gov/fgis/proficiencyprogram.aspx.
U. S. Department of Agriculture, Grain Inspections, Packers and Stockyards Administration:Grain Inspection Handbook, Book 1, Grain Sampling. Published online at http://www.gipsa.usda.gov/Publications/fgis/handbooks/gihbk1_insphb.html.
10 GENETICS OF GRAINS | Detection of Genetically Modified Grains