Special Issue: Computation and Modeling

Opinion
How Can We Better Detect
Unauthorized GMOs in Food
and Feed Chains?
Marie-Alice Fraiture,1 Philippe Herman,1 Marc De Loose,2
Frdric Debode,3 and Nancy H. Roosens1,*
Current GMO detection systems have limited abilities to detect unauthorized
Trends
genetically modied organisms (GMOs). Here, we propose a new workow,
Most European Union (EU)-unauthor-
based on next-generation sequencing (NGS) technology, to overcome this prob- ized GMOs can be detected by enfor-
lem. In providing information about DNA sequences, this high-throughput work- cement laboratories via the current
GMO detection system.
ow can distinguish authorized and unauthorized GMOs by strengthening the
tools commonly used by enforcement laboratories with the help of NGS technol- However, based on the current
ogy. In addition, thanks to its massive sequencing capacity, this workow could approach, distinguishing EU-unauthor-
ized GMOs from EU-authorized GMOs
be used to monitor GMOs present in the food and feed chain. In view of its is almost impossible.
potential implementation by enforcement laboratories, we discuss this innovative
One possible way to overcome this issue
approach, its current limitations, and its sustainability of use over time.
is a new workow using NGS technology.
The Current GMO Detection System Makes it Difcult to Detect
This new workow may improve the
Unauthorized GMOs ability of the current system to detect
To guarantee their safety and traceability in the food and feed chain as well as the freedom of EU-unauthorized GMOs.
choice for consumers, legislations regarding GMOs (see Glossary) have been established in
The high-throughput property of NGS
several countries around the world. Most of these GMO legal frameworks aim notably at
technology, enabling the generation of
regulating the introduction of GMOs into the food and feed chain. To apply for an authorized massive amounts of sequence data
introduction of a GMO, an applicant should carry out a case-by-case environmental risk from several samples in parallel, may
assessment and provide information about accurate instructions and conditions for use also enable the monitoring of GMOs that
are present in the food and feed chain.
and labeling. However, some legislation requirements, such as the labeling threshold (between
0.9% and 5%), vary among different jurisdictions. The term unauthorized GMOs concerns
1
GMOs released in the market of a certain country without prior authorization. Scientic Institute of Public Health
(WIV-ISP), Platform of Biotechnology
and Molecular Biology (PBB) and
Two main categories of unauthorized GMOs may be found in the market. First, GMOs can be Biosafety and Biotechnology Unit
considered unauthorized because they have been approved in some countries (such as the (SBB), J. Wytsmanstraat 14, 1050
Brussels, Belgium
USA, Canada, or Japan) but not yet in others [such as those in the European Union (EU)], a 2
Institute for Agricultural and Fisheries
situation referred to as asynchronous approvals, or because their time-limited regulatory Research (ILVO), Technology and
approval has expired and was not renewed [1]. However, this category of unauthorized GMOs Food Sciences Unit, Burg. Van
Gansberghelaan 115 bus 1, 9820
could be considered to be safe because they were fully characterized and are traceable on the
Merelbeke, Belgium
market using their ofcial event-specic methods. By contrast, a more problematic scenario 3
Walloon Agricultural Research Centre
can also lead to the presence of unauthorized GMOs on the market: an accidental or deliberate (CRA-W), Unit Traceability and
Authentication, Chausse de Namur
release of experimental GMOs from laboratories or eld trials (e.g., Bt10 maize, Liberty Link
24, 5030 Gembloux, Belgium
601 rice, FP967 ax, Bt63 rice, PRSV papaya, and MON71800 wheat) [2]. Unauthorized GMOs
in this category usually have not received any regulatory approval in any country, so they
*Correspondence:
could be considered to be unsafe and unknown. In addition, no or few event-specic or
nancy.roosens@wiv-isp.be
construct-specic methods are available to ensure their traceability in food and feed (N.H. Roosens).

508 Trends in Biotechnology, June 2017, Vol. 35, No. 6 http://dx.doi.org/10.1016/j.tibtech.2017.03.002

2017 Elsevier Ltd. All rights reserved.
markets. Here, the term unauthorized GMO refers in particular to this second category of Glossary
unauthorized GMOs. Construct-specic methods: real-
time PCR methods that span two
Many jurisdictions with GMO legislations apply to unauthorized GMOs a policy of zero transgenic elements inside the
transgenic cassette.
tolerance, including the EU and Japanese food and feed markets, or a policy of low DNA walking: a molecular biology
percentage threshold tolerance, such as the Russian, South Korean, and Swiss food and method based on polymerase chain
feed markets. In contrast to authorized GMOs, one of the main issues with unauthorized GMOs reactions (PCR) that can amplify
unknown sequences anking known
is that the competent authorities, such as the European Food Safety Authority (EFSA) for the EU
sequences.
market, have not assessed the biosafety of genetically modied plants and derived food and Enforcement laboratories:
feed. In addition, few or no detection methods targeting these GMOs (e.g., t35S pCAMBIA, laboratories involved in the control of
gat-tpinII, RVYK papaya, and Golden Rice 2) are available for control laboratories [316]i,ii. GMOs based on legal requirements
(Commission Regulation EC/1829/
Therefore, a workow allowing the detection of a large panel of unauthorized GMOs is crucial
2003 on GM Food and Feed, and
for safety and traceability in the food and feed chain. Commission Regulation EC/1830/
2003 on the labeling and traceability
The GMO detection workow currently used by enforcement laboratories in the EU, which of GMO). The national competent
authorities, which carry out the
uses real-time PCR (qPCR), was initially developed to target EU-authorized GMOs (Figure 1A).
sampling on market products, send
First, sequences from elements commonly found in transgenic constructs of GMOs, such as these samples to enforcement
p35S and tNOS, are targeted to cover a wide spectrum of GMOs. This screening step also laboratories for analysis. The results
usually includes makers specic to elements allowing improving the discrimination between of the analysis are then delivered to
the competent authority making the
different GMOs. This combination of screening markers varies among enforcement laborato- appropriate decisions.
ries. Given that most of the screening elements derive from natural organisms, their detection Genetically modied organism
only indicates the potential presence of GMOs in the tested samples. In many countries, such (GMO): several denitions of GMOs
as in the EU, a list of potentially identied EU-authorized GMOs is then drawn up, based on the exist. Here, GMO is dened as any
living organism that possesses a
screening analysis, to unambiguously conrm or exclude their presence. To this end, the novel combination of genetic material
corresponding GM event-specic methods are used. In addition, the GM event-specic obtained through the use of modern
methods for the authorized GM events that are not covered by the elements targeted during biotechnologyx. Generally, these
alterations comprise the insertion of
the qPCR screening are automatically tested [35,9,11,1618].
a transgenic cassette into the host
genome. The transgenic cassettes
Thus, with this system, only the presence of EU-unauthorized GMOs that have at least one are usually composed of elements
element targeted during the qPCR screening step in their transgenic cassette would be from species other than the host and
contained a gene of interest, also
suspected. However, EU-unauthorized GMOs that do not contain any of the elements targeted
known as a trait, which is highly
during the qPCR screening analysis bypass this system [35]. Nonetheless, it was recently expressed through an upstream
estimated that, by combining the screening markers specic to p35S and tNOS, approximately strong promoter and is stabilized by
90% of unauthorized GMOs are potentially covered [19]. Given that these transgenic elements a downstream terminator. The most
commonly used promoter and
are also highly represented in authorized GMOs, even if the enforcement laboratories are able
terminator are p35S and tNOS,
to detect a wide spectrum of authorized and unauthorized GMOs with the current GMO respectively.
detection system, it is not possible to discriminate between them during the screening step to GM event-specic methods: real-
determine whether the analyzed sample contains only unauthorized or authorized GMOs. In time PCR methods that can identify
unambiguously each GM event by
addition, for the subsequent identication step, no GM event-specic method is often available. targeting their unique junction
Thus, with this system, the presence of unauthorized GMOs can only be highly suspected but between the host genome and the
not proven [35,10,19,20]iii. transgenic cassette.
GTS-40-3-2 (referred as MON-
432-6): a GM soybean event
Moreover, the explanation of the screening signals by the identication of EU-authorized GMOs tolerant to herbicide developed by
could conceal the potential presence of EU-unauthorized GMOs that have similar transgenic Mosanto1. This event is one of the
elements. For example, given that the GTS-40-3-2 soybean, which contains both the p35S most encountered in the food and
feed markets worldwide. It is
and tNOS elements, is the most frequently GM event found on the worldwide market, the
authorized in many countries,
absence of discrimination is highly likely when the given sample contains both authorized and including Argentina, Australia, Brazil,
unauthorized GMOs. Therefore, the current GMO detection system is intrinsically associated Canada, China, Colombia, the EU,
with a high error rate related to the detection of unauthorized GMOs [35,10]. Japan, Korea, Malaysia, Mexico,
Paraguay, Philippines, Russia,
Singapore, South Africa, Switzerland,
To overcome these difculties, we discuss here a new workow for monitoring GMOs on the
market (Figure 1B,C). To this end, the use of the NGS technology, allowing massively parallel

Trends in Biotechnology, June 2017, Vol. 35, No. 6 509

DNA sequencing, could help to obtain the sequence information needed to prove the presence Taiwan, the USA, Uruguay, and
of EU-unauthorized GMOs in the food and feed chain (Box 1) [3,21,22]. Vietnamxi.
p35S promoter: one of the most
commonly used promoters in GMOs,
How Can We Improve the Current GMO Detection System to Better Detect derived from the cauliower mosaic
Unauthorized GMOs? virus (CaMV).
tNOS terminator: one of the most
To target a wide spectrum of GMOs, whether EU authorized or not, the qPCR screening
commonly used terminators in
analysis is retained in the new suggested workow as the initial step. This analysis targets GMOs, derived from Agrobacterium.
transgenic elements commonly found in both EU-authorized and -unauthorized GMOs, such
as p35S, tNOS, and t35S pCAMBIA. The detection of the t35S pCAMBIA element strongly
indicates the presence of EU-unauthorized GMOs in any tested samples because this element
is not found in EU-authorized GMOs and is present in approximately 30% of EU-unauthorized
GMOs. The use of additional makers specic to EU-unauthorized GMOs could also be valuable.
Unfortunately, no other elements have been selected so far due to the insufcient coverage of
EU-unauthorized GMOs. However, the detection of the p35S and/or tNOS elements only
weakly indicates the presence of EU-unauthorized GMOs because these elements are highly
present in GMOs, whether EU authorized or not [19]. The detection of these elements only
strongly indicates the presence of EU-unauthorized GMOs with samples composed exclusively
of species that do not belong to the list of EU -authorized GMOs, such as rice, wheat, or
papayaiv.

Box 1. Next-Generation Sequencing and Related Bioinformatics

NGS allows massively parallel DNA sequencing of multiple samples, which are differentiable during the subsequent
bioinformatics analysis on the basis of unique barcodes that are individually added to each sample during the library
preparation step [46]. In contrast to traditional Sanger technology, NGS offers higher throughput and substantially
reduces the need for fragment-cloning methods. The millions or billions of DNA fragments generated should then be
analyzed and interpreted with the help of bioinformatics tools [47]. Two main NGS strategies have been developed to
support a wide range of applications, including GMO detection and characterization.

On the one hand, the NGS whole-genome sequencing (WGS) approach sequences the whole DNA extracted from a
sample without any a priori knowledge of the nucleotide composition. Therefore, this NGS approach is considered to be
an untargeted strategy. The WGS approach was recently suggested as a replacement for the Southern blot in the full
molecular characterization of a single GMO in terms of its transgenic cassette(s) and its insertion sites. This application
was successfully demonstrated, for example, in the MON17903 and MON87704 soybean events, the LLRICE62, TT51-
1 and T1c-19 rice events, and the FP967 ax event [37,46,4850].

On the other hand, the NGS targeted sequencing approach refers to the sequencing of sequences of interest, isolated
earlier from the whole DNA extracted from a sample. This approach is considered to be a targeted strategy because
selecting the sequences of interest requires a minimum of knowledge. The sequences of interest could be isolated either
by PCR amplication (NGS amplicon sequencing approach) or by hybridization methods (magnetic beads or micro-
arrays) associated with specic probes (NGS trapping approach). The targeted sequencing approach has been used for
the detection and identication in the present manuscript [3,46].

The bioinformatics analysis differs according to the NGS platforms used. With the Illumina platforms, the DNA library
preparation involves the random shearing of DNA in fragments of a specic size range (i.e., 200500 bp), which can
subsequently generate sequences with specic read lengths (i.e., 100, 125, 150, or 300 bp). To properly characterize
unnatural associations of elements as well as transgene anking regions to prove the presence of GMOs in the tested
samples, the short generated DNA fragments are initially submitted during the bioinformatics analysis to a de novo
assembly analysis, comparing all generated reads to nd overlaps to rebuild some regions of the sequences. By using
the Pacic Biosciences or the Oxford Nanopore platforms, no de novo assembly step is needed. Indeed, because this
technology can deal with heterogenic libraries (DNA fragments of different sizes) and can generate long DNA fragments
(up to 60 Kbp or 200 Kbp, respectively), no shearing of the DNA library from NGS targeted approaches is required
[3,46,51].

When a new GMO sequence is observed, a verication step (i.e., PCR and Sanger sequencing) is recommended to
conrm that the sequence is not a sequencing artifact and, thus, proves the presence of a new unknown GMO in the
tested sample.

510 Trends in Biotechnology, June 2017, Vol. 35, No. 6

 Knowledge level required

High throughput

Current GMO detecon workow New suggested GMO detecon workow

(A) qPCR screening (B) qPCR screening (C) Whole-genome

Sequencing
p35S p35S
tNOS tNOS NGS trapping
...

...
+ GM event-specific methods for
authorized GMOs uncovered by the
qPCR screening

Posive signals?
Posive signals ? No GMO detected GMO detected

Posive signals? No Yes

No Yes
No GMO detected GMO detected Characterizaon of GMO sequences
using bioinformacs tools
No GMO detected GMO detected

Characterizaon by DNA walking Characterizaon

anchored on key targets by NGS trapping Signals explained by EU-authorized GMO?
Signals explained by EU-authorized GMO?
Yes No
No Yes

Signals explained by EU-authorized GMO? Idencaon and Presence of EU-

Presence of EU- Idencaon and quancaon of EU unauthorized
unauthorized GMO quancaon of EU- Yes No -authorized GMO by GMO proved
suspected authorized GMO by qPCR
qPCR
Idencaon and Presence of EU-
quancaon of unauthorized
EU-authorized GMO proved
GMO by qPCR
! Signals explained by EU-authorized GMO could
conceal the presence of EU-unauthorized GMO !

Figure 1. Current and Suggested Genetically Modied Organism (GMO) Detection Systems. (A) Current GMO detection workow used by enforcement
laboratories. Initially, the presence of authorized GMOs in a given sample is assessed by real-time polymerase chain reaction (qPCR) screening via several markers
targeting elements found in authorized GMOs (represented by p35S, tNOS, and the dotted lines). In addition, the GM event-specic methods corresponding to the
authorized GMOs uncovered by the used screening markers should be tested. Based on the signals observed during the qPCR screening, the event-specic methods
of authorized GMOs potentially detected are then tested to conrm or exclude their presence. The term No GMO detected means that no authorized GMOs as well as
no unauthorized GMOs containing the targeted sequences are observed in the tested samples. (B) Suggested workow applicable with a minimum of prior knowledge.
In the qPCR screening step, the dotted line represents the use of several screening markers. The set of screening markers is completed by the t35S pCAMBIA marker
targeting only EU-unauthorized GMOs. The term No GMO detected means that no authorized GMOs as well as no unauthorized GMOs containing the targeted
sequences are observed in the tested samples. When positive signals are observed during qPCR screening for a given sample, the potential GMOs detected are
characterized using the DNA walking or NGS trapping approaches. The generated sequences are then analyzed through bioinformatics analysis (see Box 1, main text).
The NGS trapping approach could also be used without initial qPCR screening. However, the NGS trapping approach as applied to GMOs is not currently technically
implementable in GMO routine analysis (indicated in gray). (C) Suggested workow applicable, theoretically, without any prior knowledge. Following whole-genome
sequencing of a given sample, GMOs are detected and identied using bioinformatics tools (Box 1, main text). However, this approach is not currently implementable in
GMO routine analysis.

If a positive signal is detected for key transgenic elements during the qPCR screening analysis,
a second step is then applied to obtain information about the sequences surrounding the
known transgenic elements. These sequences are subsequently compared with the ones from
EU-authorized GMOs to detect the potential presence of EU-unauthorized GMOs. To this end,
two targeted NGS strategies can currently be envisioned (Figure 1B, Box 1).

Trends in Biotechnology, June 2017, Vol. 35, No. 6 511

The rst approach, an NGS amplicon-sequencing approach, comprises a DNA walking
strategy anchored on the detected transgenic elements. The nal PCR product could then
be sequenced through NGS technologies. This kind of DNA walking approach, anchored on
the p35S, tNOS, t35S pCAMBIA, and VIP3A elements, has been successfully applied to GMO
samples, including GMOs at trace levels, GMO mixtures, and processed food matrices [13,23
28]. Alternative DNA walking strategies, described by Volpicella et al. and Arulandhu et al.,
could also potentially be used to characterize GMOs [13,19,21,29]. The generated DNA
sequences could rst be compared with a database containing at least all of the sequences
from EU-authorized GMOs to determine, in a second step, the presence or absence of EU-
unauthorized GMOs. In this way, contrary to the current qPCR GMO detection system, the
presence of EU-unauthorized GMOs would not be masked by the identication of EU-autho-
rized GMOs. Regarding the sequencing step, NGS technology can easily and substantially
increase the number of samples in one analysis via a barcoding system (Figure 2, Box 1).

Among the NGS platforms, two categories of NGS platforms can be considered. The rst,
including Illumina1, generates millions of short sequences (i.e., 100300 bp). The second,
such as the Pacic Biosciences1 and the Oxford Nanopore1 instruments, can deal with
heterogenic libraries (Box 1) and currently provides the longest read length (DNA fragments up
to 60 Kbp and 200 Kbp, respectively) [25,28] v,vi. In this way, the complexity of the bioinfor-
matics analysis could signicantly be reduced because the whole sequences of the generated
amplicons could directly be provided without any preliminary de novo assembling of short
sequences to rebuild longer sequences. These characteristics specic to the second category
of NGS platforms are also helpful for samples that comprise multiple GMOs that share some
identical transgenic elements. Additionally, experimental data have recently demonstrated the
validity of DNA walking coupled to the Pacic Biosciences platform in various types of sample
commonly found in the food and feed market [25]. However, in terms of cost, the more
conventional NGS Illumina platform represents a good alternative, as suggested by Liang et al.,
who obtained similar results with the Illumina and Pacic Biosciences technologies for analyzing
MIR162 maize event [28]. Nonetheless, additional supporting experimental data are needed for
using different GMO samples encountered in routine GMO analysis. Moreover, even if the
related bioinformatics analysis inherently requires assembling the short generated reads to
clearly observe unnatural associations of elements as well as transgene anking regions, it
could probably be simplied by developing a user-friendly bioinformatics workow for the
analysis (Box 1). Furthermore, successful implementation of this approach depends on having
a database that contains at least all of the sequences corresponding to the transgenic
cassettes and the transgene anking regions from the EU-authorized GMOs. To our knowl-
edge, no publically available database of this kind currently exists. Even if adaptations and
additional data are needed in this context, the JRC GMO-Amplicon database containing GMO
sequences generated by qPCR screening represents nowadays the rst step in this direction
[30]. Therefore, it is of utmost importance to develop an appropriate database that is as
complete as possible. However, the condentiality of certain GMO sequences could compli-
cate the accessibility of the developed database to the public.

The second option involves using the NGS trapping approach to enrich the DNA library with
sequences of interest via specic hybridization probes that capture elements commonly found
in GMOs (Box 1) [3134]. However, to our knowledge, this approach has not been applied to
GMOs, except in a preliminary experiment in the framework of a research project (UGMMO-
NITOR; convention RF 11/6242) funded by the Belgian competent authority. However, regard-
less of some encouraging results, the experiment and data analysis still need to be optimized for
application by enforcement laboratories (F. Debode, unpublished 2015). The hybridization
probes could target the screening elements as well as others sequences specic to unautho-
rized GMOs (e.g., anking regions, promoters, terminators, traits, etc.). Two important aspects

512 Trends in Biotechnology, June 2017, Vol. 35, No. 6

 Samples

...
DNA extracon

...
Library preparaon and barcoding

...
Pooling

...

Sequencing in one run

...
Demulplexing

...
Bioinformacs analysis

Comparison to databases

KeFeng6 NK603 GTS40-3-2 GTS40-3-2 NK603 GTS40-3-2 GTS40-3-2 GTS40-3-2

MON863
GTS40-3-2
NK603
MON863
KeFeng6 MON863
KeFeng6
NK603
MON863
KeFeng6
NK603 NK603
MON863 ...

Figure 2. Schematic Representation of the Targeted Next-Generation Sequencing (NGS) Approach, Based
on DNA Walking, Applied to Multiple Food and/or Feed Matrices Comprising Rice, Maize, and/or Soybean
Ingredients. After DNA extraction, it is assumed that all of these food and/or feed matrices were positive for both p35S
and tNOS in the real-time polymerase chain reaction (qPCR) screening analysis. These samples are then submitted to the
DNA walking analysis anchored on the p35S and tNOS elements. The nal PCR products from the DNA walking are then
individually barcoded for each food and/or feed matrix, represented by orange, green, light blue, red, purple, pink, or dark
blue, to be subsequently sequenced together in one run. Based on the generated sequences, earlier demultiplexed
genetically modied (GM) events, EU-authorized (e.g., NK603 maize and GTS-40-3-2 soybean) or unauthorized (e.g.,
KeFeng-6 rice and MON863 maize; underlined) genetically modied organisms can then be identied in each sample.

Trends in Biotechnology, June 2017, Vol. 35, No. 6 513

have to be taken into account. First, the use of additional hybridization probes targeting Outstanding Questions
sequences of unauthorized GMOs depends on the availability of the sequences. Second, What will be the next priorities of the
the enrichment protocol should guarantee that the captured DNA fragments of interest are long EU competent authorities regarding
unauthorized GMOs?
enough (at least >500 bp or even 1 Kbp) to characterize regions surrounding the hybridized
known sequences [35]. Even if the NGS trapping approach could be performed alone, we still Is it possible to organize a monitoring
suggest initially performing a qPCR screening to improve its effectiveness. In this way, the strategy to target unauthorized GMOs
presence of GMOs could be rapidly and inexpensively detected. Moreover, this initial step in representative samples from the EU
market through strong collaboration
avoids performing useless NGS trapping analysis with samples containing no GMOs or GMOs
between EU countries?
comprising elements untargeted by the hybridization probes. Nevertheless, given the through-
put limitation of qPCR technology, qPCR markers targeting at least elements found in a broad Are there unauthorized GMOs on the
range of GMOs, such as p35S and tNOS, should be used. For the subsequent NGS trapping market in the EU that are currently
analysis, the set of hybridization probes should at least target the elements that are revealed to undetectable?
be positive by qPCR screening.

These two NGS targeted approaches, along with several commercialized products on the EU
market, could be envisioned to monitor EU-unauthorized GMOs containing at least one
transgenic element targeted during the screening step (Figure 2).

Regarding EU-unauthorized GMOs for which the elements contained in their transgenic
construct are not targeted by the qPCR screening step, a NGS untargeted approach
may be used (Figure 1C, Box 1). To this end, the NGS whole-genome sequencing (WGS)
approach could be convenient because no a priori knowledge is theoretically required [22,36
38]. However, although this approach can be used on samples containing 100% and 10% of
a given GMO, according to a pioneering study by Willems et al., the WGS approach is
currently not applicable for samples containing GMOs only at a trace level or a mixture of
GMOs, which are both frequently encountered in routine GMO analysis [36]. For instance,
based on the statistical framework developed in this study, identifying a GM soybean at 0.1%
(per ingredient) theoretically requires generating 45 billion paired-end reads, corresponding
approximately to 20 runs with an Illumina Hiseq systemvii. Recently, the validity of this
statistical framework was also supported by experimental data generated from a maize
matrix, commercialized in the USA, in which GM maize events only with an estimated
concentration (% per ingredient) greater than or equal to 11.6% were identied. In addition,
the bioinformatics analysis could be also cumbersome for analysts without any expertise in
this eld [22,36]. Therefore, adapted tools should be developed to be accessible to enforce-
ment laboratories. Apart from these inconveniences, this WGS approach represents a
promising strategy for globally monitoring GMOs on the market, comprising the majority
of the encountered samples in GMO routine analysis, without any prior knowledge regarding
the sequences of interest.

Concluding Remarks and Future Perspectives

The new workow suggested here is proposed to strengthen the current GMO detection
system regarding unauthorized GMOs. This workow provides two main advantages. On the
one hand, the current GMO detection system used by enforcement laboratories would be
better able to detect EU-unauthorized GMOs. Indeed, EU-unauthorized GMOs, which are well
covered by the initial qPCR screening analysis, could be characterized by the sequence
information and then identied, which could unambiguously prove their presence in a given
sample. To this end, this sequence information could then be analyzed with the help of a GMO
database. With our aim of proposing a worldwide strategy, key targets, such as p35S and
tNOS, have to be present in unauthorized GMOs both from EU and non-EU countries. On the
other hand, the high-throughput property of the NGS technology used in the proposed
workow offers the opportunity to drastically increase the number of samples that could be
analyzed per experiment. In this way, monitoring unauthorized GMOs in several food and feed

514 Trends in Biotechnology, June 2017, Vol. 35, No. 6

products from the market is affordable, which is not the case with the current GMO detection
system.

Currently, biotech crops can also be produced via new plant breeding techniques [6,39].
Even if no decision has yet been taken by the EU competent authority regarding the status of
these organisms, in contrast to the USA, Australian, New Zealand, and Argentinian compe-
tent authorities, the new workow suggested here could also be used to target them,
especially by using the WGS approach for EU-unauthorized GMOs that only contain elements
untargeted during the qPCR screening analysis. However, for organisms that contain only a
small genomic modication (e.g., a change in a single or a few base pairs, or short deletions or
insertions that are not distinguishable from changes naturally occurring in plants), the
distinction between a biotech process and a conventional breeding process (e.g., crossing
and mutation techniques using chemicals or ionizing radiation) remains challenging. This kind
of biotech organism may be generated using, for example, the CRISPR-Cas9 technique. For
instance, a common white button mushroom resistant to browning, a herbicide-tolerant
maize, and a pig resistant to the porcine reproductive and respiratory syndrome virus were
recently obtained by applying the CRISPR-Cas9 approach. During early 2016, the US
competent authority ruled that this biotech mushroom was not subject to US Department
of Agriculture regulations [3944].

Therefore, due to its exible applicability for both transgenic organisms and organisms derived
from new plant breeding techniques, the workow suggested here presents many advantages
and promises sustainable use over time. Importantly, the potential implementation of the
suggested workow strongly depends on policy makers. Indeed, in the EU, routine GMO
analysis is currently performed to assess the correctness of EU-authorized GMO labeling in the
food and feed chain. However, in terms of public health and freedom of choice for consumers,
the detection of unauthorized GMOs clearly presents a higher priority because no correspond-
ing biosafety dossiers have been assessed by the competent authorities, thus representing a
potential threat to both human and animal health as well as for the environment. Therefore, to
further the efforts related to the detection of unauthorized GMOs, and to develop a plan to
monitor their presence on the market, it is important to seriously discuss this problem at the
level of the EU authorities as well as worldwide. The detection of EU-unauthorized GMOs is
already considered as a priority by the EU competent authorities. The Decision 2013/287/EU
has also been established for the detection of EU-unauthorized GM rice in the food and feed
chain. In addition, the Rapid Alert System for Food and Feed (RASFF), a key tool allowing the
rapid communication of the detection of EU-unauthorized GMOs in the food and feed chain, is
actively used in the EU [5,21] Appendix A viii,ix.

Therefore, implementing the proposed strategy in the coming years is increasingly realistic.
However, currently, its implementation in all enforcement laboratories is not practical. Indeed,
because most of the enforcement laboratories use qPCR technology, the use of a new
technology, such as NGS, will require signicant investment in terms of expertise and compu-
tational infrastructure [21,22,45]. However, the proposed strategy could be implemented
initially in only a few enforcement laboratories that could be considered as sentinels. Thus,
the GMOs present at a certain time in the food and feed chain could be monitored on a large
scale by the sentinels, instead of the current punctual sampling of a few samples over time. In
this way, the frequency of unauthorized GMOs on the market could be estimated, and the types
of food and feed product that are susceptible to containing unauthorized GMOs could be
determined (e.g., is it necessary to target all products on the market or to focus in particular on
products imported from certain countries?). Based on this information, the current system used
to control GMOs in the food and feed chain could be subjected to adaptations and measures if
necessary to improve its cost- and time-effectiveness.

Trends in Biotechnology, June 2017, Vol. 35, No. 6 515

Acknowledgment
The research that yielded these results was funded by the Belgian Federal Public Service of Health, Food Chain Safety and
Environment through the contact UGMMONITOR (convention RF 11/6242).

Resources
i
www.loc.gov/law/help/restrictions-on-gmos/restrictions-on-gmos.pdf
ii
www.gao.gov/assets/290/283060.pdf
iii
www.gmoseek.com/gmoseek
iv
http://gmo-crl.jrc.ec.europa.eu/emerg-unauth.html
v
https://nanoporetech.com/
vi
www.pacb.com/smrt-science/smrt-sequencing/read-lengths/
vii
www.illumina.com/content/dam/illumina-marketing/images/systems/miniseq/takeover/PDF/
brochure-sequencing-systems-portfolio.pdf
viii
https://ec.europa.eu/food/sites/food/les/safety/docs/oc_eurl_wp_2016_gmo_en.pdf
ix
http://ec.europa.eu/food/safety/rasff_en
x
https://bch.cbd.int/protocol/text/
xi
www.cera-gmc.org/GmCropDatabaseEvent/GTS%2040-3-2

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