detection of genetically modified...

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.


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.


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.


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).


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


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?


• 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

Bravo A, Gill SS, and Sobero M (2007) Mode of action of Bacillus thuringiensis Cry andCyt toxins and their potential for insect control. Toxicon 49: 423–435.

CLI, www.detection-methods.com (accessed 19 February 2015).

Dixon LK and Hohn T (1985) Cloning and manipulating Cauliflower Mosaic Virus.In: Becker Y and Hadar J (eds.) Recombinant DNA Research and Viruses.Developments in Molecular Virology, Vol. 5. pp. 247–275.

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.

Saiki R, et al. (1988) Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase. Science 239: 487–491. http://dx.doi.org/10.1126/science.2448875.

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.


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