2 GMO Holst Jensen

8/13/2019 2 GMO Holst Jensen

1/12

Research review paper

Testing for genetically modied organisms (GMOs): Past, present andfuture perspectives

Arne Holst-Jensen

Department of Feed and Food Safety, National Veterinary Institute, Ullevaalsveien 68, P.O. Box 750 Sentrum, 0106 Oslo, Norway

a b s t r a c ta r t i c l e i n f o

Available online 27 May 2009

Keywords:

Transgenic crops

Detection

Identication

Quantication

Transformation

Quality assurance

High throughput

Validation

Unapproved GMO

Intragenics

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve

in the next decade. Challenges and limitations for the application of the test methods as well as to the

interpretation of results produced with the methods are highlighted and discussed, bearing in mind the

various interests and competences of the involved stakeholders. To better understand the suitability and

limitations of detection methodologies the evolution of transformation processes for creation of GMOs is

briey reviewed.

2009 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10712. Stakeholder roles and responsibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072

3. Development and evolution of GMOs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073

4. Method development and availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076

5. Unauthorised GMOs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077

6. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078

1. Introduction

Gene technology as a technology is potentially a short way to

improving domesticated plants and animals, mainly because it can

bypass biological barriers for recombination and genetic exchange

across unrelated species by creating transgenes. However, from a

societal point of view this technology is seen by many as a derail

rather than a short way, as many still do not feel that the safety of the

technology is conclusively demonstrated. Like any other technology,

the gene technology has a potential for hostile abuse or unsafe use.

Because of its wide ranging potential, the public perception of the

technology has also affected the regulation of the technology and the

testing requirements; to an extent more comparable to medical

drugs than to any other food related production technology, at least in

some parts of the world.

Therst commercial genetically modied (GM) plant (the FlavrSavr

tomato) was authorised for marketing in 1994 (Food and Drug

Administration, 1994). As will become evident in the following, it was

more future oriented than the majority of the currently marketed rst

generation GM organisms(GMOs) in some ways.The term GMOmay be

used in a broad sense to include all life forms, but the most common

application of the term is limited to conne GM plants and animals. In

the following, GMOs will refer strictly to GM plants because GM animals

are yet to be commersialised, except for ornamental sh and pets. The

annual increase in commercial plantings of GMOs has risen with an

average of approximately 10% over the last decade. In 2007 GMOs

occupied more than 143 million ha in 23 countries, with soybean, cotton,

maize and rapeseed (canola) as the dominant crops (James, 2008).

Regulation of gene technology varies from country to country. A

few issues are fairly common, however: assessments on a case-by-

Biotechnology Advances 27 (2009) 10711082

Tel.: +47 2321 6243; fax: +47 2321 6202.

E-mail address:[email protected].

0734-9750/$ see front matter 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.biotechadv.2009.05.025

Contents lists available at ScienceDirect

Biotechnology Advances

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.biotechadv.2009.05.025http://www.sciencedirect.com/science/journal/07349750http://www.sciencedirect.com/science/journal/07349750http://dx.doi.org/10.1016/j.biotechadv.2009.05.025mailto:[email protected]


2/12

case basis, with focus on safety, a distinction between contained use

and release into the environment, and a distinction between growing

and using (e.g. as raw material for food or feed incl. processing). The

concept of substantial equivalence (to the non-GMO isogenic counter-

part) is widely adopted as a basis for assessing a GMO ( Schauzu,

2000). A GMO may become deregulated (e.g. USA), may become

authorised for limited use, such as imports as food (e.g. the EU), or

specic products derived from specic GMOs may become authorised

(e.g. PR China). Tolerance thresholds (e.g. for non-authorised GMOs)or thresholds for labeling are in place in some but not all countries.

Specic thresholds also vary and labeling thresholds maybe voluntary

or mandatory. As a consequence, the specic needs for detection,

identication and quantitation vary signicantly.

Testing for (detection of) GMOs may serve several purposes.

Qualitative testing may be used to discriminate between authorised

and unauthorised material or use of material, to identify safe or

potentially unsafe material, or for certication of purity of identity

preserved material. Quantitative testing may be used to control for

compliance with legal (e.g. for labeling) or contractually agreed thresh-

olds (e.g. with respect to botanical impurity). Testing may also play a

role in the safety assessment and risk management of GMOs by

providing a means of tracing and if necessary retracting the GMO

material, by providing data from characterisation of the GMO itself

(see e.g. Collonnier et al.,2005; Hernandez et al.,2004; Taverniers et al.,

2005; Windels et al., 2001, 2003) and from environmental samples

(Aono et al., 2006; Messean et al., 2007; Ortiz-Garcia et al., 2005; Quist

and Chapela, 2001; Saji et al., 2005). Testing may also provide tools to

implement traceability of GMO derivatives in animals and humans

that have consumed GM material (Chowdhury et al., 2003a,b; Nielsen

et al., 2005).

2. Stakeholder roles and responsibilities

The stakeholders (users of tests) and the analysts (providers of

tests) play distinct roles and may have very diverse competence and

interests in the GMO testing. Trust and reliability are keywords. Inac-

curate test reports may be misleading, but reporting also has a

translation component since the analytical competence of the stake-holders is often inferior and certainly differs from that of the analyst.

Misperception and inaccuracy may lead to incorrect or suboptimal

decisions by the stakeholders. The test report therefore must provide

information not only about the test result but also about the uncer-

tainties and limitations associated with the test result. This informa-

tion must be presented in a form that is perceived and interpreted

correctly by the stakeholder. The responsibilities of the analysts

include: 1) appropriate choice of testing method, including method

validation status; 2) identication of potential sources of error in

reporting and translation of results; and 3) communication with the

stakeholdersa priori, explaining what the analyst can provide, and a

posteriori, explaining what the results mean including relevant

limitations. Most testing is not performed by the same people who

sample the material that is subject to testing, and sampling is notcovered in the present paper. Because the sampling error may be

much larger than the analytical measurement uncertainty or error, the

interested reader is referred to Allnutt et al., (2008); Brera et al.,

(2005); Bridges, (2007); Committe Europeen de Normalisation (CEN),

(2006);Degrieck et al., (2005);Emslie et al., (2007);Kobilinsky and

Bertheau, (2005); Paoletti et al., (2006); and Remund et al., (2001) for

more information on sampling.

Selecting the analytical method can be challenging when multiple

optional methods are available. The best choice for one laboratory or

situation is not necessarily the best for another. Minimizing cost and

time are often prioritized, but this can easily be at the expense of

reliability. The European Network of GMO Laboratories (ENGL) has

prepared a guidance document for method acceptance (European

Network of GMO Laboratories, 2008) that has been adopted by the

European Commission's Community Reference Laboratory for GM

Food and Feed (CRL-GMFF). The document is biased towards quantita-

tive real-time PCR methods. This and other related documents on the

CRL-GMFF website http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm

may be useful for analytical laboratories in selecting, comparing and

validating methods. If a multilaboratory trial has been performed on a

method, it is often easier to assess its reliability than if the validation

of the method is limited to experiments performed by a single (devel-

oping) laboratory. Method harmonisation is often desirable because itfacilitatestransparency and comparison of results between laboratories.

Norms or standards have been published or are under establishment

at regional and national level in many countries (e.g. P.R. China, France,

Germany, Korea, Japan, Switzerland) as well as at European and inter-

national level (Codex Alimentarius Commission, 2008; International

Organization for Standardization, 2004, 2005a,b,c,d, 2006). Interna-

tional method harmonisation is reinforced by the publication of collab-

orative trial validated methods and validation reports by the CRL-GMFF

(European Commission, 2009). However, there may also be reasons to

choose non-harmonised methods. Cost, specicity, convenience, new

information or availability of reference materials are examples.

Recently, a GMO database (Dong et al., 2008) providing informa-

tion on detection methods and including specic DNA sequences of

inserted and anking elements in many of the GMOs, was launched.

This database, although still suffering from minor inaccuracies in

sequence annotations and lacking considerable parts of inserts of

several GMOs, will certainly be a useful tool for method developers

and even more so for analytical laboratories and stakeholders who

wish to interpret or verify test results. Contributing to update this

database and possibly establish mirror versions in other countries,

could be seen as a collective task for stakeholders involved in detec-

tion of GMOs.

Uncertainty and error associated with a measurement result must

be communicated in the test reports. The analyst must identify the

potential sources of uncertainty and error, and quantify or at least

establish a relative rank of the contribution by each source (Zel et al.,

2007). Lack of correspondence between test results produced in dif-

ferent laboratories may be caused by differences in methods speci-

city, sensitivity or analyte recovery, resulting in bias. However,several biological factors are too often ignored by analysts and stake-

holders (Holst-Jensen et al., 2006). The unit of measurement and

expression of GMO content is often indicated as the mass:mass ratio

of GMO:ingredient, partly because the rst generation of certied

GMO testing reference materials were mass based (Trapmann et al.,

2002a,b). However, the analytical methods applied usually measure

particular analytes such as the number of copies of DNA molecules or

proteins (Holst-Jensen et al., 2003, 2006).

In order to facilitate translation from DNA based to mass based

concentration of GMO, it has been proposed that a conversion factor

may be applied (Kuribara et al., 2002; Shindo et al., 2002). The factor

may adjust for some of the most inuential biological factors

discussed by Holst-Jensen et al., 2006. However, the proposed

conversion factor is always derived from the reference material usedand does not necessarily reect the nature of the sample material. So,

if for example the reference material is derived from a hemizygous

GMO while the sample is derived from a homozygous GMO, then the

conversion factor will yield an estimated mass based concentration

that is approx. twice the true mass based concentration.

Cloned DNA was therefore early proposed as an attractive and t-

for-purpose alternative type of reference material (Blockand Schwarz,

2003; Burns et al., 2006; Kuribara et al., 2002; Shindo et al., 2002;

Taverniers et al., 2001, 2004). The Japanese GMO standards have

applied cloned DNA reference materials since 2002 (Shindo et al.,

2002). More recently, reference materials have been made available

certied for their DNA analyte content (Charels et al., 2007a,b). For

qualitative purposes, and when the reference material is particularly

precious, it may be suitable to use whole genome amplied DNA from

1072 A. Holst-Jensen / Biotechnology Advances 27 (2009) 10711082
http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htmhttp://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm


3/12

genomic DNA samples (Roth et al., 2008). However, this type of

reference material is clearly unt for quantitation.

The materials analysed in the laboratory may vary from low pro-

cessed grains to highly processed composite foods or feeds. The

quality and quantity of analyte extracted and puried from the sam-

ples may vary correspondingly (Cankar et al., 2006; Moreano et al.,

2005). The laboratory should always report the limits of detection

(LOD) and/or quantication (LOQ) together with the test result. It is,

however, important to distinguish between an LOD/LOQ determinedfor the analytical method and the LOD/LOQ determined for the sample

subject to analysis. A processed and composite product may contain

very little of the target analyte compared to an unprocessed single

ingredient product. Consequently, the LOD/LOQ of the sample may be

100-fold inferior to the LOD/LOQ of the method (Berdal et al., 2008;

Berdal and Holst-Jensen, 2001; Holst-Jensen et al., 2003). In such

cases, reporting only the method specic LOD/LOQ is misleading to

the stakeholders reading the test report, and may easily give an

unjustied positive impression of the reliability of a negative result.

3. Development and evolution of GMOs

The rst GMOs on the market were plants carrying single trait

genes regulated by only a few common promoter (mainly the cauli-

ower mosaic virus 35S promoter) and terminator (mainly the

Agrobacterium tumefaciens3nos) elements (AgBios, 2008; Hemmer,

1997). The transformation vectors carried an additional marker gene

construct, usually transcriptionally inactive in the plant. With few

exceptions, the traits were agronomic, dominated by herbicide

(glyphosate, gluphosinate and oxinyl) tolerance and insect resistance

(various forms ofBacillus thuringiensis Cry proteins). For laboratories

wishing to develop detection methods, availability of corresponding

reference material was often a problem (Holst-Jensen et al., 2003).

Consequently, the GMO testing methods developed were focused on

detection of generic elements or features found in the GMOs such as

the promoter and terminator elements (DNA based methods) or Cry

proteins in their native form (Anklam et al., 2002; Hernandez et al.,

2005a,b; Holst-Jensen et al., 2003; James et al., 2003; MacCormick

and Grifn, 1998; Rodriguez-Lazaro et al., 2007). As the number of

commercialised GMOs increased towards the turn of the millennium,it became evident that the generic (screening) methods were often

unable to comply with stakeholder requirements such as authorisa-

tion and labelling regulations because identication and/or quantita-

tion could not be achieved.

With a few early exceptions such as the Flavr Savr tomato, which

was ripening delayed giving enhanced shelf life, practically all com-

mercialised GMOs express agronomic traits. Traits primarily benecial

to theconsumers arestillin thepipeline, despite optimistic announce-

ments from developers over the last decade (Engel et al., 2002).

Examples include rice enriched with vitamin A and altered fatty acid

composition of oils from oleaceous grains (AgBios, 2008).

A new trend began with the new millennium, where multiple

agronomic traits were combined (gene stacking or pyramiding; see

Taverniers et al., 2008and references therein). This introduced a new

challenge to the analysts. With exception for testing of single seeds or

tissue derived from individual plants, none of the existing detection

methods could discriminate between the combined presence of two

or more single trait GMOs and stacked GMOs (Akiyama et al., 2005;

Holst-Jensen et al., 2006; Taverniers et al., 2008). The number of traits

that have been combined in stacked GMOs is rapidly growing, and the

rst 8-stack (SmartStax) is presently subject to regulatory review by

Fig. 1.Evolution of GMO detection methods and associated reference materials.

1073A. Holst-Jensen / Biotechnology Advances 27 (2009) 10711082


4/12

Table 1

Examples of PCR based detection methods.a

Species Event OECD unique ID Tradename(s) Construct C, geneG

or event EspecicbReference

Cotton 281-24-236 DAS-24236-5 Widestrike E Baeumler et al. (2006),European Commission (2009)

3006-210-23 DAS-21023-5 Widestrike E Baeumler et al. (2006),European Commission (2009)

GBH614 BCS-GH002-5 E European Commission (2009)

GK12 C Cheng et al. (2007)

GK19 C Yang et al. (2005a)

LLCOTTON25 ACS-GH001-3 E European Commission (2009)ME (QL) Kim et al. (2008b)

Mon531 MON-00531-6 C Yang et al. (2005a)

E European Commission (2009),Yang et al. (2005b)

Mon1445 MON-01445-2 E European Commission s(2009),Yang et al. (2005b)

ME (QL) Kim et al. (2008b)

Mon15985 MON-15985-7 BollGardII C Lee et al. (2007)

E European Commission (2009)


Mon88913 MON-88913-8 C Lee et al. (2007)


SGK321 C Yang et al. (2005a)

Maize (corn) 3272 SYN-E3272-5 E European Commission (2009)

59122 DAS-59122-7 E European Commission (2009)

Bt10 E (QL) European Commission (2009),Watanabe et al. (2007)

Bt11 SYN-BT011-1 C Brodmann et al. (2002),Matsuoka et al. (2002),

Peano et al. (2005b)

MC (QL) Germini et al. (2004),Hernandez et al. (2003b),

Matsuoka et al. (2001a),Onishi et al. (2005),

Peano et al. (2005a)

MC Rudi et al. (2003)

E (comp) Zimmermann et al. (2000)

E European Commission (2009),Rnning et al. (2003),

Taverniers et al. (2005)

ME (QL) Hernandez et al. (2005a,b),Xu et al. (2007)

Bt176 SYN-EV176-9 G Bordoni et al. (2004),Ehlers et al. (1997)

G (comp) Garcia-Canas et al. (2004a,b)

C Brodmann et al. (2002),Matsuoka et al. (2002),

Peano et al. (2005b),Vatilingom et al. (1999)

MC (QL) Germini et al. (2004),Hernandez et al. (2003b),




E Taverniers et al. (2005)

ME (QL) Xu et al. (2007)

CBH351 Starlink MG Rudi et al. (2003)C Matsuoka et al. (2001b)s

E (QL) Windels et al. (2003)

DBT418 MG Rudi et al. (2003)

DLL25 C Matsuoka et al. (2002)

Event 32 DAS-59132-8 E European Commission (2009)

GA21 MON-00021-9 MG Rudi et al. (2003)

C Hernandez et al. (2004),Matsuoka et al. (2002),


MC (QL) Germini et al. (2004),Hernandez et al. (2005a,b),

Hernandez et al. (2003b),Matsuoka et al. (2001a),

Nadal et al. (2006),Onishi et al. (2005),Peano et al.

(2005a)

E European Commission (2009),Oguchi et al. (2008),

Taverniers et al. (2005)


LY038 REN-00038-3 C

E European Commission (2009)Mir604 SYN-IR604-5 C

E European Commission (2009)

Mon802 C Matsuoka et al. (2002)

Mon810 MON-00810-6 MaisGard C Brodmann et al. (2002),Matsuoka et al. (2002),


C (comp) Zimmermann et al. (1998)

MC (QL) Germini et al.(2004),Hernandez et al. (2003b),




E European Commission (2009),Gasparic et al.

(2008),Hernandez et al. (2003a),Holck et al.

(2002),Huang and Pan (2004),La Paz et al.

(2007),Moreano et al. (2006),Pang et al. (2007),

Salvi et al. (2008)



5/12

Table 1(continued)


or eventEspecicbReference

ME (QL) Hernandez et al. (2005a,b),Huang and Pan

(2004),Nadal et al. (2006),Xu et al. (2007)

Mon863 MON-00863-5 C Lee et al. (2006)

MC (QL) Onishi et al. (2005)

E European Commission (2009),Pan et al. (2006),

Yang et al. (2005d)


Mon88017 MON-88017-3 E European Commission (2009)

Mon89034 MON-89034-3 E European Commission (2009)

NK603 MON-00603-6 MC (QL) Onishi et al. (2005)

E European Commission (2009),Huang and Pan

(2004),Nielsen et al. (2004)

ME (QL) Huang and Pan (2004),Nadal et al. (2006)

T25 C Brodmann et al. (2002),Matsuoka et al. (2002)

MC (QL) Matsuoka et al. (2001a),Onishi et al. (2005)


E Collonnier et al. (2005),European Commission

(2009),Papazova et al. (2006)

ME (QL) Hernandez et al. (2005a,b),Xu et al. (2007)

TC1507 DAS-01507-1 MC (QL) Onishi et al. (2005)

E European Commission (2009),

La Paz et al. (2006)

Papaya 55-1 (63-1) C Lo et al. (2007),Wall et al. (2004),Yamaguchi et al. (2006)

Pepper Non-authorised

herbicide tolerant

C Song et al. (2007)

Potato Bt6 New leaf C Rho et al. (2004),Watanabe et al. (2004)

EH92-527-1 BPS-25271-9 E Broothaerts et al. (2007),European Commission (2009)

RBMT21-350 New leaf plus C Rho et al. (2004),Watanabe et al. (2004)

SEMT15-15 New leaf Y C Rho et al. (2004),Watanabe et al. (2004)

E Watanabe et al. (2004)

Non-authorised

fungal resistant ac2

G Pribylova et al. (2006)

Rapeseed (canola) Falcon GS40/90 ACS-BN-1-4 Liberty link C Block and Schwarz (2003)

GT73 MON-00073-7 MC (QL) Kim et al. (2007)

E European Commission (2009),Kim et al. (2006a),

Taverniers et al. (2005),Yang et al. (2007)

ME (QL) Demeke and Ratnayaka (2008)

Ms1 ACS-BN004-7 E Wu et al. (2007)

Ms8 ACS-BN005-8 MG (QL) Demeke and Ratnayaka (2008)

MC (QL) Kim et al. (2007)

E European Commission (2009),Wu et al. (2008)

OXY-235 ACS-BN011-5 MC (QL) Demeke and Ratnayaka (2008)Rf1 ACS-BN001-4 E Wu et al. (2007)

Rf2 ACS-BN002-5 E Wu et al. (2007)

Rf3 ACS-BN003-6 MG (QL) Demeke and Ratnayaka (2008)

MC (QL) Kim et al. (2007)

E European Commission (2009),Wu et al. (2008)

T45 ACS-BN008-2 MC (QL) Kim et al. (2007)

E European Commission (2009),Yang et al. (2006)

ME (QL) Demeke and Ratnayaka (2008)

Topas 19/2 ACS-BN007-1 E Wu et al. (2009)

Rice Bt63 C European Commission (2009),Mde et al. (2006)

LL62 ACS-OS002-5 E European Commission (2009)

LL601 E (QL) European Commission (2009)

Soybean A2704-12 ACS-GM005-3 E European Commission (2009)

A5547-127 ACS-GM006-4 E European Commission (2009)

DP-305423-1 DP-305423-1 E European Commission (2009)

DP -356043-5 DP-35604 3-5 European Commission (2009)

GTS40-3-2 MON-04032-6 RoundupReady MG (QL) Dainese et al. (2004)

C Kim et al. (2004),Lerat et al. (2005) Liu et al.(2005),Pan and Shih (2003),Peano et al. (2005b),

Tani et al. (2005),Vatilingom et al. (1999),

Vollenhofer et al. (1999),Wang and Fang (2005),

Zhang et al. (2007),Zhou et al. (2007)

DC Foti et al. (2006)

MC Germini et al. (2004),Hernandez et al. (2003b),


E Berdal and Holst-Jensen (2001),Burns et al. (2003),

European Commission (2009),Huang and Pan (2005),

Moreano et al. (2006),Pang et al. (2007),Taverniers

et al. (2001),Terry and Harris (2001)


MON89788 MON-89788-1 E European Commission (2009)

Squash CZW-3 C Wall et al. (2004)

ZW-20 C Wall et al. (2004)

(continued on next page)



6/12

the US Environmental Protection Agency (EPA) and is expected to

reach the market in 2010 (http://www.news.dow.com/dow_news/

corporate/2008/20080616a.htm). There is good reason to anticipate

that this trend will continue. However, in stead of combining single

agronomic trait genes, the future GMOs may be transformed with

gene clusters for example encoding partial or complete synthetic

pathways. Articial and engineered chromosomes can be developed

and allow forcombining the desired genes on stable inheritable single

chromosomes (Birchler et al., 2008). This can be achieved in maize by

exploiting the short supernumerary B chromosome to engineer a

minichromosome comprised of little more than a centromere and a

recombination segment where the insert(s)can be added. This system

potentially improves genetic control and traceability, and may

facilitate gene dosage regulation compared to Agrobacterium

mediated or biolistic transformation techniques.

Some stakeholders have expressed concern about the safety andethics of transgenics, viz. moving genes across species barriers

(Nielsen, 2003; Russel and Sparrow, 2008). Gene technology may

also be applied to intragenicsand famigenics, i.e. to move genes within

or between reproductively compatiblespecies, essentially speeding up

conventional breeding processes (Nielsen, 2003). This could raise

fewer concerns, but the diversity of available and attractive trait genes

would be drastically reduced. Interestingly, therst commercial GMOs

were intragenics; the Flavr Savr tomato from Calgene and a similar

tomato from Zeneca were modied by insertion of a truncated version

of a tomato polygalacturonase gene (AgBios, 2008). Silencing of

undesirable genes via intragenics is another option that may reduce

safety and ethics concern (Weeks et al., 2008).

Although there has been impressive progress towards the devel-

opment of synthetic living organisms (SLOs) (Lartigue et al., 2007),there is still a gap between the transfer of a copied genome to a new

host and introduction of a designed novel genome ( Holt, 2008). This

technology will undoubtedly raise a cascade of ethical and scientic

debates. While contained use of SLO microorganisms may be

considered a realistic scenario within 1 or 2 decades, extension to

environmental release is unlikely to obtain societal acceptance in the

foreseeable future. Advanced life formssimilar to plants or animals are

almost beyond imagination.

4. Method development and availability

The tools applied for GMO testing (Fig. 1) are primarily bioassays,

protein based (mainly immunological) assays and DNA based assays

(mainly applying the polymerase chain reaction [PCR] technology).

However, also other technologies have been developed and these will

briey be reviewed and discussed in the following along with the

dominant technologies.

Testing for a single trait or GMO event may require only a simple

method, whereas testing for presence of multiple events, possibly for

identication and quantication may require use of combinations of

methods (Christianson et al., 2008; Holst-Jensen, 2007; James et al.,

2003; Waiblinger et al., 2008).

Bioassays are based on the principle of exposing plant seedlings

from a seed batch to e.g. a herbicide to which the GMO plants are

tolerant whereas non-GMOs are susceptible. Counting the survivors

and compare with the number of affected plants will give the relative

GMO content in the seed lot. The advantages of bioassays are poten-

tially low costs, few requirements for user competence and the ability

of the assays to conrm the desired biological properties of the GMOs.

Their drawback is that they can only be applied to certain biologicalproperties, they usually require longer time to perform than protein

and DNA based assays, and their specicity is limited.

Proteins can be detected by application of immunological and

physicochemical techniques. The most common protein based assays

are immunoassays where the target proteins (the antigens) are detec-

ted by specic antibodies coupled to a colorimetric detection system.

Application of immunoassays has evolved little (Fig. 1) compared to

application of DNA based methods, and they are mainly applied as

convenient and cost effective screening tools for the large scale

farming industry working with single ingredient materials of low or

unprocessed grade. Two main factors may partially account for the

limited evolution: the costs of developing specic antibodies, and the

fact that antibodies can not be described and synthesised in a simple

way in contrast to oligonucleotides applied with DNA based methods.Signicant improvements are mainly a shift from polyclonal to more

specic monoclonal antibodies, and from laboratory based enzyme

linked immunosorbent assays (ELISA) to portable lateral ow strips

(LFS) that can be applied in the eld, at on- and off-loading points and

at storage and processing facilities. The potential for quantitation has

also improved. Although multiplexing of immunological methods

could be achieved using microarray formats (Ling et al., 2007), the

most promising initiative towards multiplexing of immunoassays for

GMO detection until now is one involving the application of coloured

beads coated with the antibodies and analysed by ow cytometry

(Fantozzi et al., 2007).

Because the expression and translation of genes can be low, e.g. if

regulated by a tissue specic promoter or affected by environmental

factors, sensitivity and reliable quantiability is often a problem with

Table 1(continued)


or event EspecicbReference

Sugarbeet RUR H7 KM-000H71-4 E European Commission (2009)

Tomato FlavrSavr C Meyer (1995)

Huafan no1. C Yang et al. (2005c)

Wa termelon Non-au thor ised

virus resistant

G Kim et al. (2008a)

Unspecic t axon Scr eening

marker (gene)

Target version Originally applied to

3nos various GMOs Liu et al. (2007),Reiting et al. (2007),Sun et al. (2007)

Cry1A(b) GM maize Danson et al. (2006)

Cry1A(c) GM cotton Cheng et al. (2007),Singh et al. (2007)

Cry1Ab (Bt176) Lutz et al. (2006)

Cry1Ba GM maize Danson et al. (2006)

Cry9C CBH351 Starlink maize Orlandi et al. (2002),Quirasco et al. (2004)

P35S Various GMOs Cankar et al. (2008),Kunert et al. (2006),Liu

et al. (2007),Xu et al. (2006a)

Pat/bar Chinese cabbage Lim et al. (2007)

Pat/pat T25 maize Weighardt et al. (2004)

Vip3A/vip-s Cotton and tobacco Singh et al. (2008)

a This table does not list multiplex methods where the majority of targets are typical screening targets.b MC=multiplex method where this target is construct specic, MG =multiplex method where this target is gene specic, ME =multiplex method where this target is event

specic. QL=method is qualitative but not quantitative.

http://www.news.dow.com/dow_news/corporate/2008/20080616a.htmhttp://www.news.dow.com/dow_news/corporate/2008/20080616a.htmhttp://www.news.dow.com/dow_news/corporate/2008/20080616a.htmhttp://www.news.dow.com/dow_news/corporate/2008/20080616a.htm


7/12

immunoassays. Improved sensitivity may be achieved for example

by combining the immunoassay with PCR (Allen et al., 2006). Genes

regulated by constitutive promoters may also have relatively stable

expression and translation levels. Quantitative application of immu-

noassays is an option under certain conditions but normally not in

highly processed or composite products (Ermolli et al., 2006;

Rodriguez-Nogales et al., 2008; Shim et al., 2007; Van den Bulcke

et al., 2007; van Duijn et al., 2002).

Alternative protein based methods described include use of immu-nomagnetic electrochemical sensors (Volpe et al., 2006), 2-dimen-

sional gel electrophoresis (Kim et al., 2006b) and mass spectrometry

(Ocana et al., 2007).

DNA based assays are particularly applied by global traders, the food

processingindustry and law enforcement authorities. The advantagesof

DNA based assays are primarily specicity and sensitivity. Their

drawbacks are primarily cost and competence requirements. Since the

genetic modication bydenition aremodications of DNA, it is evident

that DNA based methods are at the highest level of metrological

traceability, compared to methods that detect and measure transcrip-

tional (RNA), translational (protein) or phenotypic (bioassays) deriva-

tives of the modied DNA, respectively (listed in falling order of

metrological traceability).

Table 1is an incomplete list of major GMOs and PCR based meth-

ods for their detection. The Table includes multiplex methods and the

validation status for many of the methods is uncertain. It is strongly

recommended to make further investigations to ensure that only

validated methods are applied for diagnostic purposes. One of the

most sensational and debated studies of the environmental impact of

GMOs (Quist and Chapela, 2001) has been heavily criticized for

possible use of insufciently validated methods or methods unt-for-

purpose and lack of appropriate controls (Christou, 2002; Kaplinsky

et al., 2002; Metz and Ftterer, 2002) with the consequence that the

results are subject to reduced acceptance.

A wide range of alternativesto conventionalgel electrophoresisexist

fordetection and identicationof thePCR amplied targets: capillarygel

electrophoresis (Garcia-Canas et al., 2004b; Heide et al., 2008a,b; Nadal

et al., 2006), hybridisation to labelled and coloured beads and ow

cytometry (Fantozzi et al., 2008), array hybridisation (Germini et al.,2005; Hamels et al., 2009; Leimanis et al., 2006; Morisset et al., 2008a;

Prins et al., 2008; Xu et al., 2007, 2006b), immunological detection with

dipsticks (Kalogianni et al., 2006), surface plasmon resonance (Feriotto

et al., 2003), various electrochemical sensors (Kumar and Kang, 2007;

Sun et al., 2007, 2008; Xu et al., 2006a) and detection by liquid chroma-

tography and mass spectrometry (Shanahan et al., 2006).

While PCR is the dominating DNA based technology, alternatives

such as isothermal amplication (Fukuta et al., 2004; Morisset et al.,

2008a,b), direct detection of genomic DNA by electrochemical sensors

(Stobiecka et al., 2007), cDNA analysis by microarray (Chen et al., 2004)and direct hybridisation of genomic DNA to microarrays (Nagarajan and

De Boer, 2003; Tengs et al., 2007) have also been proposed.

High throughput methods have recently been developed and are

presently nding their way to routine laboratories. These tools

become particularly attractive if they can be combined with automa-

tion technology. These high throughput methods are either based on

combinations of one or several oligoplex PCRs followed by multiplex

(pooled) identication of the amplied DNA (Hamels et al., 2009;

Heide et al., 2008a,b; Leimanis et al., 2008; Mano et al., 2009; Nadal

et al., 2006) or apply multiple simultaneous PCRs (Chaouachi et al.,

2008; Mano et al., 2009).

5. Unauthorised GMOs

One of the main challenges of today is the possible presence of

unauthorised GMO or derived material in the food chain or the

environment (Holst-Jensen, 2008; U.S. Department of Agriculture,

2008; United States Government Accountability Ofce, 2008; Vermij,

2006). There is currently a rapid increase in the number of GMO events

authorised in some but not all countries (sometimes referred to as

asynchronous authorisation) and the number of additional events

subject to eld trials potentially leading to pollenow (Fox, 2003, 2001;

Holst-Jensen, 2008; Krueger and Le Buanec, 2008; MacCormick and

Grifn, 1998). Illegal intended or unintended releases for example from

experiments in research laboratories mayalso takeplace,although with

a very low probability (Holst-Jensen, 2008). These may pose signicant

risks to human and animal health and the environment.

The immediate consequences are affecting international trade

(European Commission, 2007; Krueger and Le Buanec, 2008; U.S.Department of Agriculture, 2008; United States Government

Fig. 2.The matrix approach. An example of a stepwise application. 1) A relational matrix correlating the performance of specic analytical modules with known GMO events is rst

created. It is recommended to verify experimentally that the correlation is true. If a module is known to yield a positive signal when reference material of a particular GMO event is

analysed, then a +is tabulated in the matrix (upper left). If a module is known to yield a negative signal, then a is tabulated. 2) Samples are analysed using either a multiplex

method or multiple simplex modules for screening targets A, B, C, D and E, and construct speci c targets F, G and H. The results for each sample and target is tabulated (lower left).

3) The observed patterns for each sample are matched against the relational matrix. If all targets known to yield a positive signal with reference material of a particular event are

positive when thesample is tested, then thematchis Perfect forthatparticular GMOeventin that particular sample(upper right). If themajority butnot allof thetargetsknownto

yield a positive signalwithreference materialare positive,then thematch is Part_missing. Ifonly a minority of thetargets knownto yield a positive signalwith referencematerialis

positive, then the match is Mostly_missing. If none of the targets known to yield a positive signal with reference material are positive, then the match isNegative. 4) To check for

possible presence ofGMO eventsnot includedin therelationalmatrix,e.g.unauthorisedGMOevents,the matchpatternis further examined,e.g.listing allobservedtargetsthatcanbe

referred to one or more Perfectmatches, followed by a slash and a list of the targets that cannot be referred to one or more Perfectmatches (lower right). The latter list of targets

maybe used asa basis forassessmentof presence ofmaterialfroman unauthorisedGMO event.Notably, thematrixapproachmaybe appliedin a numberof differentbutrelatedways.

Usually,a softwareis neededto support datainterpretation.Vericationmaybe done,e.g.usingeventspecic analyticalmodules.When concentrationsof targets arenear or belowthe

limit of detection, then the probability of false negatives is high, and this may affect the match scoring. Care should therefore always be taken when results are interpreted.



8/12

Accountability Ofce, 2008). Other possible consequences include

reduced consumer trust in the industry, technology and authorities.

For the incidents of unauthorised events reported so far, no evidence of

signicant harm to human healthhas been provided.Yet, these repeated

incidents challenge the present regulations in many countries that

require authorisationbasedon thorough safetyassessments prior to any

release or marketing. To cope with the challenge, a number of detection

approaches have been developed and additional approaches are under

development. The simplest include application of event speci

cmethods for unauthorised events (Akiyama et al., 2007; Babekova

et al., 2009; Grohmann and Mde, 2009; Mde et al., 2006 ). Another

alternative is application of combinations of screening methods and

comparing the results with tabulated data on presence/absence in

individual authorised events; where patterns that do not match are

indicative of presence of unathorised GMO (James et al., 2003; Mano

et al., 2009; Waiblinger et al., 2008). This approach is often referred to as

the matrix approach (Chaouachi et al., 2008; Hamels et al., 2009;

Remacle and Bertheau, 2001) (Fig. 2) and is favoured by many

laboratories as part of their general GMO screening strategy e.g. prior

to application of quantitative and conrmative event specic detection.

One of the drawbacks of the matrix approach is that it does not provide

conclusive evidence of the presence of unauthorised GMO. It is also

hampered by the uncertain performance of the screening tests with

individual GMOs, and may be challenged by simultaneous presence of

multiple GMOs in a sample. One strategy that may at least partially

improve theapplicability of thematrix approach is theuse of differential

quantitative screening (Cankar et al., 2008). Combining screening

targets with ngerprinting approaches such as anchor PCR (Theuns

et al., 2002) followed by conrmatory sequencing of the suspected

amplied fragment(s) may further facilitate the detection and identi-

cation of unauthorised GMOs (Taverniers et al., pers.commun.), as

exemplied by the detection of unexpected inserts and rearranged DNA

in Roundup Ready soybean by (Windels et al., 2001). However, this

approach requires a comprehensive fragment prole reference database

and may not be suited for samples with low level presence. Finally,

advanced approaches based on screening of whole genomes for foreign

DNA e.g. by microarray analysis have been proposed and partly

demonstrated (Nesvold et al., 2005; Tengs et al., 2007).

6. Future perspectives

Current testing methods are generally too expensive and unt for

in-eld applications, or lack sufcient specicity and ability to

quantify the GMOs. Future technologies are expected to be faster,

cheaper and allow for both multiplexing and quantitation, should be

portable and yet sufciently specic and quantitative. Achieving all

these goals may be difcult. In the meantime it is likely that

laboratories will be increasingly dependent on efcient screening

strategies based on both multiplex protein and DNA screening,

applying a matrix approach to determine the need for more specic

identication and quantication methods. Semiquantitative methods

may nd increased popularity when formal thresholds are in place, inorder to sort samples into three categories: 1) compliant with specic

quantitative threshold, 2) non-compliant with specic threshold, and

3) in need for further quantitative analysis.

Low level presence of events that are not authorised but have

been evaluated and considered safe in other countries may become

more acceptable in many countries in order to minimise negative

effects on global trade. However, on a global scale the requirements

for GMO testing with particular focus on presence of unauthorised

GMOs are likely to increase, see e.g. (U.S. Department of Agriculture,

2008; United States Government Accountability Ofce, 2008). Given

the limitations of the matrix approach as currently implemented, it

is evident that there will be a need for developments of new

analytical tools to facilitate detection of non-tolerable unauthorised

events.

Novel transformation technologies may redene the types

of analytical target. Intragenics, SNPs and gene silencing to some extent

preclude the use of protein or PCR based GMO detection. Possible

alternatives to cope with these include genome microarrays, DNA se-

quencing approaches, electrochemical sensors and mass spectrometry.

International harmonisation and exchange of information on GMO

developments,eldtrials andDNA sequencesinvolved, at least among

competent authorities and their associated control laboratories, may

not only facilitate monitoring for unauthorised GMOs, but could alsobe a necessity to reduce possible negative consequences for global

trade. Finally, developments in regulation of biotechnology and

requirements for authorisation and labeling may result in altered

priorities and create new challenges to the GMO testing laboratories,

as exemplied by the present challenges associated with gene stacks

(Taverniers et al., 2008).

References

AgBios. GMO database; 2008.http://www.agbios.com/dbase.php.Akiyama H, Watanabe T, Wakabayashi K, Nakade S, Yasui S, Sakata K, et al. Quantitative

detection system for maize sample containing combined-trait genetically modiedmaize. Anal Chem 2005;77(22):74218.

Akiyama H, Sasaki N, Sakata K, Ohmori K, Toyota A, Kikuchi Y, et al. Indicated detectionof two unapproved transgenic rice lines contaminating vermicelli products. J Agric

Food Chem 2007;55(15):5942

7.Allen RC, Rogelj S, Cordova SE, Kieft TL. An immuno-PCR method for detecting BacillusthuringiensisCry1Ac toxin. J Immunol Methods 2006;308(12):10915.

Allnutt T, Dwyer M, McMillan J, Henry C, Langrell S. Sampling and modeling for thequantication of adventitious genetically modied presence in maize. J Agric FoodChem 2008;56(9):32327.

Anklam E, Gadani F, Heinze P, Pijnenburg H, Van den Eede G. Analytical methods fordetection and determination of genetically modied organisms in agriculturalcrops and plant-derived food products. Eur Food Res Technol 2002;214(1):326.

Aono M, Akiyama S, Nagatsu M, NakajimaN, Tamaoki M, Kubo A, et al.Detectionof feraltransgenic oilseed rape with multiple herbicide resistance in Japan. EnvironBiosafety Res 2006;5:7787.

BabekovaR, Funk T,PecoraroS, Engel K, Busch U.Developmentof an event-specic real-time PCR detection method for the transgenic Bt rice line KMD1. Eur Food ResTechnol 2009;228(3):70716.

Baeumler S, Wulff D, Tagliani L, Song P. A real-time quantitative PCR detection methodspecic to widestrike transgenic cotton (event 281-24-236/3006-210-23). J AgricFood Chem 2006;54:652734.

Berdal KG, Holst-Jensen A. Roundup Ready soybean event-specic real-time quanti-

tative PCR assay and estimation of the practical detection and quanti cation limitsin GMO analyses. Eur Food Res Technol 2001;213:4328.

Berdal KG, Bydler C, Tengs T, Holst-Jensen A. A statistical approach for evaluation ofPCR results to improve the practical limit of quantication (LOQ) of GMO analyses(SIMQUANT). Eur Food Res Technol 2008;227:114957.

Birchler J, Yu W, Han F. Plant engineered minichromosomes and articial chromosomeplatforms. Cytogenet Genome Res 2008;120:22832.

Block A, SchwarzG. Validation of different genomic and cloned DNAcalibration standardsfor construct-specic quantication of LibertyLink in rapeseed by real-time PCR.Eur Food Res Technol 2003;216(5):4217.

Bordoni R, Mezzelani A, Consolandi C, Frosini A, Rizzi E, Castiglioni B, et al. Detection andquantitation of genetically modied maize (Bt-176 Transgenic maize) by applyingligationdetectionreactionand universal arraytechnology. J Agric FoodChem 2004;52(5):104954.

Brera C, Donnarumma E, Onori R, Foti N, Pazzaglini B, Miraglia M. Evaluation of samplingcriteria for the detection of gm soybeans in bulk. Ital J Food Sci 2005;17(2):17785.

Bridges, A. (2007). Sampling and detection methods for products of modern agri-cultural biotechnology in NAFTA countries. International Life Sciences Institute(ILSI) International Food Biotechnology Committee.

Brodmann P, Ilg E, Berthoud H, Herrmann A. Real-time quantitative polymerase chainreaction methods for four genetically modied maize varieties and maize DNAcontent in food. J AOAC Int 2002;85(3):64653.

Broothaerts W, Corbisier P, Emons H, Emteborg H, Linsinger T, Trapmann S. Develop-ment of a certied reference material for genetically modied potato with alteredstarch composition. J Agric Food Chem 2007;55:472834.

Burns M, Shanahan D, Valdivia H, Harris N. Quantitative event-specic multiplex PCRdetection of Roundup Ready soya using LabChip technology. Eur Food Res Technol2003;216(5):42833.

Burns M, Corbisier P, Wiseman G, Valdivia H, McDonald P, BowlerP, et al.Comparison ofplasmid and genomic DNA calibrants for the quantication of genetically modiedingredients. Eur Food Res Technol 2006;224(2):24958.

Cankar K, Stebih D, Dreo T, Zel J, Gruden K. Critical points of DNA quantication by real-time PCR effects of DNA extraction method and sample matrix on quanticationof genetically modied organisms. BMC Biotechnol 2006;6.

Cankar K, Chauvency-Ancel V, Fortabat MN, Gruden K, Kobilinsky A, Zel J, et al. Detectionof nonauthorized genetically modied organisms using differential quantitativepolymerase chain reaction: application to 35S in maize. Analy Biochem2008;376(2):

189

99.

http://www.agbios.com/dbase.phphttp://www.agbios.com/dbase.php


9/12

Chaouachi M, Chupeau G, Brard A, McKhann H, Romaniuk M, Giancola S, et al. A high-throughput multiplex method adapted for GMO detection. J Agric Food Chem2008;56(24):11596606.

Charels D, Broeders S, Corbisier P, Trapmann S, Schimmel H, Emons H. Toward metrol-ogical traceability for DNA fragment ratios in GM quantication. 3. Suitability ofDNA calibrants studied with a MON 810 corn model. J Agric Food Chem 2007a;55(9):326874.

Charels D, Broeders S, Corbisier P, Trapmann S, Schimmel H, Linsinger T, et al. Towardmetrological traceability for DNA fragment ratios in GM quantication. 2. Systematicstudy of parameters inuencing the quantitative determination of MON 810 corn byreal-time PCR. J Agric Food Chem 2007b;55(9):325867.

Chen TL, Prasad V, Lee CH, Lin KH, Chiueh LC, Chan MT. Extending the cDNA microarraydetection system to evaluate genetically modied soybean and traditional soyfoods. J Food Drug Anal 2004;12(3):25965.

Cheng H, JinW, WuH, Wang F,YouC, Peng Y,et al.Isolation andPCR detectionof foreignDNA sequences in bee honey raised on genetically modied Bt (Cry1Ac) cotton.Food Bioprod Process 2007;85(C2):1415.

ChowdhuryEH, Kuribara H,Hino A, SultanaP,Mikami O,Shimada N,et al.Detectionof cornintrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinalcontentsof pigsfed genetically modied cornBt11. J AnimSci 2003a;81(10):254651.

Chowdhury EH, Shimada N, Murata H, Mikami O, Sultana P, Miyazaki S, et al. Detectionof Cry1Ab protein in gastrointestinal contents but not visceral organs of geneticallymodied Bt11-fed calves. Vet Hum Toxicol 2003b;45(2):725.

Christianson J, McPherson M, Topinka D, Hall L, Good A. Detecting and quantifying theadventitious presence of transgenic seeds in safower,Carthamus tinctorius L. J AgricFood Chem 2008;56(14):550613.

ChristouP. No crediblescientic evidenceis presentedto support claims thattransgenicDNAwas introgressed into traditional maize landraces in Oaxaca, Mexico. Transgenic Res2002;11(1):35.

Codex Alimentarius Commission. Codex ad hoc intergovernmental task force on foods

derived from biotechnology; 2008. http://www.who.int/foodsafety/biotech/codex_taskforce/en/.

Collonnier C, Schattner A, Berthier G, Boyer F, Coue-Philippe G, Diolez A, et al. Charac-terization and event specic-detection by quantitative real-time PCR of T25 maizeinsert. J AOAC Int 2005;88(2):53646.

Committe Europeen de Normalisation (CEN).TS 15568 FoodstuffsMethodsof analysisforthe detection of genetically modied organisms and derived productsSamplingstrategies; 2006. p. 15568.

Dainese E, Angelucci C, De Santis P,MaccarroneM, Cozzani I. A multiplex PCR-based assayfor the detection of genetically modied soybean. Anal Lett 2004;37(6):113950.

Danson JW, Kimani M, Mbogori M. Detection ofBacillus thuringiensis genes in trans-genic maize by the PCR method and FTA paper technology. Afr J Biotechnol 2006;5(22):23459.

DegrieckI, Silva ED,Van Bockstaele E, De Loose M. QuantitativeGMO detectionin maize(Zea mays L.) seed lots by means of a three-dimensional PCR based screeningstrategy. Seed Sci Technol 2005;33(1):3143.

Demeke T, Ratnayaka I. Multiplex qualitative PCR assay for identication of geneticallymodied canola events and real-time event-specic PCR assay for quantication ofthe GT73 canola event. Food Control 2008;19(9):8937.

Dong W, Yang L, Shen K, Kim B, Kleter G, Marvin H, et al. GMDD: a database of GMOdetection methods. BMC Bioinformatics 2008;9:260.

EhlersB, Strauch E,GoltzM, KubschD, Wagner H,MaidhofH, etal. NachweisgentechnischerVernderungen in Mais mittels PCR. Bundesgesundheitsblatt 1997;97(4):11821.

Emslie K, Whaites L, Grifths K, Murby E. Sampling plan and test protocol for thesemiquantitative detection of genetically modied canola (Brassica napus) seed inbulk canola seed. J Agric Food Chem 2007;55(11):441421.

Engel K, Frenzel T, Miller A. Current and future benets from the use of GM technologyin food production. Toxicol Lett 2002;127(13):32936.

Ermolli M, Prospero A, Balla B, Querci M, Mazzeo A, Van den Eede G. Developmentof aninnovative immunoassay for CP4EPSPS and Cry1AB genetically modied proteindetection and quantication. Food Addit Contam 2006;23(9):87682.

European Commission. Economic impact of unapproved GMOs on EU feed imports andlivestockproduction.Directorate-General for Agriculture and Rural Development; 2007.

European Commission. Status of dossiers; 2009. http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm.

European Network of GMO Laboratories. Denition of minimum performancerequirements for analytical methods of GMO testing; 2008. http://gmo-crl.jrc.ec.

europa.eu/doc/Min_Perf_Requir_Analyt_methods_131008.pdf.Fantozzi A, Ermolli M, Marini M, Scotti D, Balla B, Querci M, et al. First application of a

microsphere-based immunoassay to the detection of genetically modied organ-isms (GMOs): quantication of Cry1Ab protein in genetically modied maize.

J Agric Food Chem 2007;55(4):10716.Fantozzi A, Ermolli M, Marini M, Balla B, Querci M, Van den Eede G. Innovative appli-

cation ofuorescent microsphere based assay for multiple GMO detection. FoodAnal Methods 2008;1(1):107.

Feriotto G, Gardenghi S, Bianchi N, Gambari R. Quantitation of Bt-176 maize genomicsequences by surface plasmon resonance-based biospecic interaction analysis ofmultiplex polymerase chain reaction (PCR). J Agric Food Chem 2003;51(16):46406.

Food and Drug Administration (1994). First biotech tomato marketed. FDA ConsumerSeptember 1994. US Food and Drug Administration (FDA), Center for Food Safetyand Applied Nutrition (CFSAN).

Foti N, Onori R, Donnarumma E, Santis B, Miraglia M. Real-time PCR multiplex methodfor the quantication of Roundup Ready soybean in raw material and processedfood. Eur Food Res Technol 2006;222(12):20916.

Fox JL. EPA re-evaluates StarLink license. Nat Biotechnol 2001;19(1):11.Fox JL. Puzzling industry response to ProdiGene asco. Nat Biotechnol 2003;21(1):34.

Fukuta S, Mizukami Y, Ishida A, Ueda J, Hasegawa M, Hayashi I, et al. Real-time loop-mediated isothermal amplicationfor theCaMV-35S promoter as a screening methodfor genetically modied organisms. Eur Food Res Technol 2004;218(5):496500.

Garcia-Canas V, Cifuentes A, Gonzalez R. Quantitation of transgenic Bt event-176 maizeusing double quantitative competitive polymerase chain reaction and capillary gelelectrophorsesis laser-induced uorescence. Anal Chem 2004a;76(8):230613.

Garcia-Canas V, Gonzalez R, Cifuentes A. Sensitive and simultaneous analysis of vetransgenic maizes using multiplex polymerase chain reaction, capillary gel electro-phoresis, and laser-induced uorescence. Electrophoresis 2004b;25(14):221926.

Gasparic M, Cankar K, Zel J, Gruden K. Comparison of different real-time PCR chemistriesandtheir suitabilityfor detection andquanticationof geneticallymodied organisms.

BMC Biotechnol 2008;8:26.Germini A, Zanetti A, Salati C, Rossi S, Forre C, Schmid S, et al. Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean andmaize in feeds and foods. J Agric Food Chem 2004;52(11):327580.

Germini A, Rossi S, Zanetti A, Corradini R, Fogher C, Marchelli R. Development of apeptide nucleic acid array platform for the detection of genetically modiedorganisms in food. J Agric Food Chem 2005;53(10):395862.

Grohmann L, Mde D. Detection of genetically modied rice: collaborative trialvalidation study of a construct-specic real-time PCR method for detection oftransgenic Bt rice. Eur Food Res Technol 2009;228(3):497500.

Hamels S, Glouden T, Gillard K, Mazzara M, Debode F, Foti N, et al. A PCR-microarraymethod for the screening of genetically modied organisms. Eur Food Res Technol2009;228(4):53141.

Heide B, Drmtorp S, Rudi K, Heir E, Holck A. Determination of eight genetically modiedmaize events by quantitative, multiplex PCR and uorescence capillary gel electro-phoresis. Eur Food Res Technol 2008a;227(4):112537.

Heide B, Heir E, Holck A. Detection of eight GMO maize events by qualitative, multiplexPCRanduorescencecapillarygel electrophoresis.Eur Food Res Technol 2008b;227(2):52735.

Hemmer W. Foods derived from genetically modied organisms and detection methods.BATS-Report 2/97, BATS, Basel, Switzerland; 1997.

Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A. A speci c real-timequantitative PCR detection system for event MON810 in maize YieldGard (R) basedon the 3-transgene integration sequence. Transgenic Res 2003a;12(2):17989.

Hernandez M, Rodriguez-Lazaro D, Esteve T, Prat S, Pla M. Development of meltingtemperature-based SYBR Green I polymerase chain reaction methods for multi-plex genetically modied organism detection. Analy Biochem 2003b;323(2):16470.

Hernandez M, Esteve T, Prat S, Pla M. Development of real-time PCR systems based onSYBR Green I, Ampliuor and TaqMan technologies for specic quantitativedetection of transgenic maize event GA21. J Cereal Sci 2004;39(1):99107.

Hernandez M, Rodriguez-Lazaro D, Ferrando A. Current methodology for detection,identication and quantication of geneticallymodiedorganisms. Curr Anal Chem2005a;1:20321.

Hernandez M, Rodriguez-Lazaro D, Zhang D, Esteve T, Pla M, Prat S. Interlaboratorytransfer of a PCR multiplex method for simultaneous detection of four geneticallymodied maize lines: Bt11, MON810, T25, and GA21. J Agric Food Chem 2005b;53(9):33337.

Holck A, Va M, Didierjean L, Rudi K. 5-nuclease PCR for quantitative event-specicdetection of the genetically modied Mon810 MaisGard maize. Eur Food Res Technol2002;214(5):44953.

Holst-Jensen A. Sampling, detection, identication and quantication of geneticallymodied organisms (GMOs). In: Pico Yolanda, editor. Food Toxicants Analysis.Amsterdam: Elsevier; 2007. p. 23168.

Holst-Jensen A. GMO testing: trade, labeling or safety rst? Nat Biotechnol 2008;26(8):8589.

Holst-Jensen A, Rnning SB, Lvseth A, Berdal KG. PCR technology for screening andquantication of genetically modied organisms (GMOs). Anal Bioanal Chem2003;375:98593.

Holst-Jensen A, De Loose M, Van den Eede G. Coherence between legal requirementsand approaches for detection of genetically modied organisms (GMOs) and theirderived products. J Agric Food Chem 2006;54:2799809.

Holt R. Synthetic genomes brought closer to life. Nat Biotechnol 2008;26(3):296 7.Huang HY, Pan TM. Detection of genetically modied maize MON810 and NK603 by

multiplexand real-time polymerase chainreaction methods.J AgricFoodChem 2004;52(11): 32648.

Huang CC, Pan TM. Event-specic real-time detection and quantication of geneticallymodied Roundup Ready soybean. J Agric Food Chem 2005;53(10):38339.

International Organization for Standardization. 21572:2004 Foodstuffs Methods ofanalysis for the detection of geneticallymodied organisms and derived productsprotein based methods. Geneva, Switzerland: International Organization forStandardization (ISO); 2004. EN ISO 21572:2004.

International Organization for Standardization. TS 21098:2005 FoodstuffsNucleic acidbased methods of analysis for the detection of genetically modied organisms andderived productsinformation to be supplied and procedure for the addition ofmethods to ISO 21569, ISO 21570and ISO 21571. Geneva, Switzerland: InternationalOrganization for Standardization; 2005a. ISO/TS 21098:2005.

International Organization for Standardization. 21571:2005 FoodstuffsMethods ofanalysis for the detection of genetically modied organisms and derived productsnucleic acid extraction. Geneva, Switzerland: International Organization forStandardization (ISO); 2005b. EN ISO 21571:2005.

International Organization for Standardization. 21570:2005 FoodstuffsMethods ofanalysis for the detection of genetically modied organisms and derived productsquantitative nucleic acid based methods. Geneva, Switzerland: InternationalOrganization for Standardization; 2005c. EN ISO 21570:2005.

http://www.who.int/foodsafety/biotech/codex_taskforce/en/http://www.who.int/foodsafety/biotech/codex_taskforce/en/http://gmo/http://gmo/http://gmo/http://gmo/http://www.who.int/foodsafety/biotech/codex_taskforce/en/http://www.who.int/foodsafety/biotech/codex_taskforce/en/


10/12

International Organization for Standardization. 21569:2005 FoodstuffsMethods of ana-lysis for the detection of genetically modied organisms and derived productsqualitative nucleic acid based methods. Geneva, Switzerland: International Organiza-tion for Standardization; 2005d. EN ISO 21569:2005.

International Organization for Standardization. 24276:2006 FoodstuffsMethods ofanalysis for the detection of genetically modied organisms and derived productsgeneral requirements and denitions. Geneva, Switzerland: International Organi-zation for Standardization (ISO); 2006. EN ISO 24276:2006.

James C. Global status of commercializedbiotech/GM crops: 2007. ISAAA Brief 37 2008,International Service for the Acquisition of Agri-Biotech Applications (ISAAA); 2008.

James D, Schmidt AM, Wall E, Green M, Masri S. Reliable detection and identication of

genetically modied maize, soybean, and canola by multiplex PCR analysis. J AgricFood Chem 2003;51(20):582934.Kalogianni DP, Koraki T, Christopoulos TK, Ioannou PC. Nanoparticle-based DNA bio-

sensor for visual detection of genetically modied organisms. Biosens Bioelectron2006;21(7):106976.

Kaplinsky N, Braun D, Lisch D, Hay A, Hake S, Freeling M. Maize transgene results inMexico are artefacts. Nature 2002;416:601.

Kim YT, Park BK, Hwang EI, Yim NH, Lee SH, Kim SU. Detection of recombinant markerDNA in genetically modied glyphosate-tolerant soybean and use in environmentalrisk assessment. J Microbio Biotechnol 2004;14(2):3904.

Kim JH, Song HS, Kim DH, Kim HY. Quantication of genetically modied canola GT73using TaqMan real-time PCR. J Microbio Biotechnol 2006a;16(11):177883.

KimVH, ChoiSJ, LeeHA, MoonTW.Quantitation ofCP4 5-enolpyruvyishikimate-3-phosphatesynthase in soybean by two-dimensional gel electrophoresis. J Microbio Biotechnol2006b;16(1):2531.

Kim JH, Kim TW, Lee WY, Park SH, Kim HY. Multiplex PCR detection of the GT73,MS8xRF3, and T45 varieties of GM canola. Food Sci Biotechnol 2007;16(1):1049.

Kim CG, Lee B,KimDI, ParkJE,KimHJ,ParkKW, etal. Detection ofgeneowfrom GMtonon-GM watermelon in a eld trial. J Plant Biol 2008a;51(1):747.

Kim J, Kim S, Seo Y, Lee W, Park S, Kim H. Multiplex PCR detection of the MON1445,MON15985,MON88913, and LLcotton25varieties of GM cotton. Food Sci Biotechnol2008b;17(4):82932.

Kobilinsky A, Bertheau Y. Minimum cost acceptance sampling plans for grain control,with application to GMO detection. Chemometr Intell Lab Syst 2005;75(2):189200.

Krueger R, Le Buanec B. Action neededto harmonize regulation of low-level presence ofbiotech traits. Nat Biotechnol 2008;26(2):1612.

Kumar KS, Kang SH. Ultra-fast simultaneous analysis of genetically modied organismsin maize by microchip electrophoresis with LIF detector. Electrophoresis 2007;28(22):424754.

Kunert R, GachJS, Vorauer-UhlK, Engel E, Katinger H. Validated method forquanticationof gentically modied organisms in samplesof maize our. J Agric FoodChem2006;54(3):67881.

Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, et al. Novel referencemolecules for quantitation of genetically modied maize and soybean. J AOAC Int2002;85(5):107789.

La Paz JL, Garcia-Muniz N, Nadal A, Esteve T, Puigdomenech P, Pla M. Interlaboratorytransfer of a real-time polymerasechain reaction assayfor quantitative detection ofgenetically modied maize event TC-1507. J AOAC Int 2006;89(5):134752.

LaPaz JL,Esteve T,Pla M. Comparison ofreal-time PCRdetection chemistries andcyclingmodes using Mon810 event-specic assays as model. J Agric Food Chem 2007;55(11):43128.

Lartigue C, Glass J, Alperovich N, Pieper R, Parmar P, Hutchison C, et al. Genome trans-plantationin bacteria:changingone species toanother. Science 2007;317(5838):6328.

Lee SH, Min DM, Kim JK. Qualitative and quantitative polymerase chain reaction analysisfor genetically modied maize MON863. J Agric Food Chem 2006;54(4):11249.

LeeSH, KimJK, Yi BY. Detectionmethods forbiotechcotton MON15985 andMON 88913by PCR. J Agric Food Chem 2007;55(9):33517.

Leimanis S, Hernandez M, Fernandez S, Boyer F, Burns M, Bruderer S, et al. A microarray-based detection system forgenetically modied (GM) foodingredients. Plant MolBiol2006;61(12):12339.

Leimanis S, Hamels S, Naze F, Mbella G, Sneyers M, Hochegger R, et al. Validation of theperformance of a GMO multiplex screening assay based on microarray detection.Eur Food Res Technol 2008;227(6):162132.

Lerat S, England LS, Vincent ML, Pauls KP, Swanton CJ, Klironomos JN, et al. Real-timepolymerase chain reaction quantication of the transgenes for roundup ready corn

and roundup ready soybean in soil samples. J Agric Food Chem 2005;53(5):1337

42.Lim C, Kim S, Choi Y, Park YD, Kim SU, Sung SK. Utilization of the bar gene to develop

an efcient method for detection of the pollen-mediated gene ow in Chinesecabbage (Brassica rapaspp. pekinensis). Plant Biotechnol Rep 2007;1(1):1925.

Ling M, Ricks C, Lea P. Multiplexing molecular diagnostics and immunoassays usingemerging microarray technologies. Expert Rev Mol Diagn 2007;7(1):8798.

Liu YC, Lin SH, Lai HM, Jeng ST. Detection of genetically modi ed soybean and itsproduct tou-kan by polymerase chain reaction with dual pairs of DNA primers. EurFood Res Technol 2005;221(6):72530.

Liu GM, Cai HN, Cao MH, Su WJ. Fluorophore double stranded probe-multiplex quan-titative PCR method for detecting transgenic component of promoter derived fromCauliower Mosaic Virus and nos terminator derived from Agrobacterium tume-

facienssimultaneously. Food Control 2007;18(6):61522.Lo CC, Chen SC, Yang JZ. Use of real-time polymerase chain reaction (PCR) and

transformation assay to monitor the persistence and bioavailability of transgenicgenes released from genetically modied papaya expressing nptII and PRSV genesin the soil. J Agric Food Chem 2007;55(18):753440.

Lutz B, WiedemannS, Albrecht C. Degradationof transgenicCry1Ab DNAand protein in Bt-176maize during the ensilingprocess.J AnimPhysiol AnimNutr 2006;90(34):11623.

MacCormickC, Grifn HUHGM.Common DNA sequenceswithpotential fordetection ofgenetically manipulated organisms in food. J Appl Microbiol 1998;84(969):980.

Mde D, Degner C, Grohmann L. Detection of genetically modied rice: a construct-specic real-time PCR method based on DNA sequences from transgenic Bt rice.Eur Food Res Technol 2006;224(2):2718.

Mano J, Shigemitsu N, Futo S, Akiyama H, Teshima R, Hino A, et al. Real-time PCR arrayas a universal platform for the detection of genetically modied crops and itsapplication in identifying unapproved genetically modied crops in Japan. J AgricFood Chem 2009;57(1):2637.

Matsuoka T, Kuribara H, Akiyama H, Miura K , Goda Y, Kusakabe Y, et al. A multiplex PCRmethod of detecting recombinant DNAs from ve lines of genetically modied

maize. J Food Hyg Soc Jpn 2001a;42(1):24

32.Matsuoka T, Kuribara H, Suefuji S, Miura K, Kusakabe Y, Akiyama H, et al. A detectionmethod for recombinant DNA from genetically modied maize CBH351. J Food HygSoc Jpn 2001b;42(3):197201.

Matsuoka T, Kuribara H, Takubo K, Akiyama H, Miura K, Goda Y, et al. Detection ofrecombinant DNA segments introduced to genetically modied maize. J Agric FoodChem 2002;50:21009.

Messean A, Sausse C, Gasques J, DarmencyH. Occurrence of genetically modied oilseed rapeseeds in the harvest of subsequent conventional oilseed rape over time. Eur J Agron2007;21:11522.

Metz M, Ftterer J. Suspectevidence of transgenic contamination.Nature 2002;416:6001.Meyer R. Nachweis gentechnologisch vernderter Panzen mittels der Polymerase

Kettenreaktion (PCR) am Beispiel derFlavr SavrTM-Tomate. Z Lebensm Forsch A Eur Food Res Technol 1995;201:5836.

Moreano F, Busch U, Engel KH. Distortion of genetically modied organism quantica-tion in processed foods: Inuence of particle size compositions and heat-inducedDNA degradation. J Agric Food Chem 2005;53(26):99719.

Moreano F, Ehlert A, Busch U, Engel KH. Ligation-dependent probe amplication for thesimultaneous event-specic detection and relative quantication of DNA from two

genetically modied organisms. Eur Food Res Technol 2006;222(56):47985.Morisset D, Dobnik D, Hamels S, Zel J, Gruden K. NAIMA: target amplication strategy

allowing quantitative on-chip detection of GMOs. Nucleic Acids Res 2008a;36:e118.Morisset D, Stebih D, Cankar K, Zel J, Gruden K. Alternative DNA ampli cation methods

to PCR and their application in GMO detection: a review. Eur Food Res Technol2008b;227(5):128797.

Nadal A, Coll A, La Paz JL, Esteve T, Pla M. A new PCR-CGE (size and color) method forsimultaneousdetectionof geneticallymodied maizeevents. Electrophoresis 2006;27:387988.

Nagarajan MM, De Boer SH. An oligonucleotide array to detect genetically modi edevents in potato. Plant Mol Biol Report 2003;21(3):25970.

Nesvold H, Kristoffersen A, Holst-Jensen A, Berdal K. Design of a DNA chipfor detection ofunknown genetically modied organisms (GMOs). Bioinformatics 2005;21:191726.

Nielsen K. Transgenic organisms time for conceptual diversication? Nat Biotechnol2003;21(3):2278.

Nielsen C, Berdal K, Holst-Jensen A. Characterisation of the 5 integration site anddevelopment of an event-specic real-time PCR assay for NK603 maize from a lowstarting copy number. Eur Food Res Technol 2004;219:4217.

Nielsen C, Berdal K, Bakke McKellep A, Holst-Jensen A. Dietary DNA in blood and liverofAtlantic salmon (Salmo salarL.). Eur Food Res Technol 2005;221:18.

Ocana MF, FraserPD, Patel RKP, Halket JM,BramleyPM. Mass spectrometric detectionofCP4 EPSPS in genetically modied soya and maize. Rapid Commun Mass Spectrom2007;21(3):31928.

Oguchi T, Onish M, Chikagawa Y, Minegishi Y, Kodama T, Akiyama H, et al. Developmentof event-specic quantitationmethod forGA21 maize,whichis a GM event withoutCaMV35S promoter. J Food Hyg Soc Jpn 2008;49(1):1622.

Onishi M, Matsuoka T,KodamaT, KashiwabaK, Futo S, Akiyama H, et al.Developmentofa multiplex polymerase chain reaction method for simultaneous detection of eightevents of genetically modied maize. J Agric Food Chem 2005;53(25):971321.

Orlandi PA, Lampel KA, South PK, Assar SK, Carter L, Levy DD. Analysis ofour and foodsamples for cry9Cfrom Bioengineered Corn. J Food Prot 2002;65(2):42631.

Ortiz-GarciaS, Ezcurra E, SchoelB, Acevedo F,SobernonJ, Snow A. Absenceof detectabletransgenes in local landraces of maize in Oaxaca, Mexico (20032004). Proc NatlAcad Sci U S A 2005;102(35):1233843.

PanTM, ShihTW. Detection of genetically modied soybeansin misoby polymerasechainreaction and nested polymerase chain reaction. J Food Drug Anal 2003;11(2):1548.

Pan AH, Yang LT, Xu SC, Yin CS, Zhang K W, Wang ZY, et al. Event-specic qualitative and

quantitative PCR detection of MON863 maize based upon the 3-transgene

integration sequence. J Cereal Sci 2006;43(2):2507.Pang S, Qureshi F, Shanahan D, Harris N. Investigation of the use of rolling circle

amplication for thedetection of GM food. EurFood ResTechnol 2007;225(1):5966.Paoletti C, Heissenberger A, Mazzara M, Larcher S, Grazioli E, Corbisier P, et al. Kernel lot

distributionassessment(KeLDA):a study on thedistribution of GMOin large soybeanshipments. Eur Food Res Technol 2006;224(1):12939.

Papazova N, Taverniers I, Degrieck I, Van Bockstaele E, Joost H, De Loose M. Real timepolymerase chain reaction (PCR) quantication of T25 maize seeds inuence ofthe genetic structures in the maize kernel on the quantitative analysis. Seed SciTechnol 2006;34(2):30717.

PeanoC, BordoniR,GulliM,Mezzelani A,Samson MC,De BellisG, etal. Multiplexpolymerasechain reaction and ligation detection reaction/universal array technology for thetraceability of genetically modied organisms in foods. Analy Biochem 2005a;346(1):90100.

Peano C, Lesignoli F,GulliM, CorradiniR, SamsonMC, Marchelli R, et al.Developmentofa peptide nucleic acid polymerase chain reaction clamping assay for semiquanti-tative evaluation of genetically modied organism content in food. Analy Biochem2005b;344(2):17482.



11/12

Pribylova R, Pavlik I, Rozsypalova Z, Bartos M. A PCR-based method for the detection ofgenetically modied potatoes by the geneac2fromAmaranthus caudatus. Eur FoodRes Technol 2006;223(1):13942.

Prins T, Van Dijk J, Beenen H, Van Hoef A, Voorhuijzen M, Schoen C, et al. Optimisedpadlock probe ligation and microarray detection of multiple (non-authorised)GMOs in a single reaction. BMC Genomics 2008;9:584.

Quirasco M, Schoel B, Plasencia J, Fagan J, Galvez A. Suitability of real-time quantitativepolymerase chain reaction and enzyme-linked immunosorbent assay for cry9Cdetection in Mexican corn tortillas: Fate of DNA and protein after alkaline cooking.

J AOAC Int 2004;87(3):63946.Quist D, Chapela I. Transgenic DNA introgressed into traditional maize landraces in

Oaxaca, Mexico. Nature 2001;414(6863):541

3.Reiting R, Broll H, Waiblinger HU, Grohmann L. Collaborative study of a T-nos real-timePCR method for screening of genetically modied organisms in food products.

J Verbrauch Lebensm 2007;2:11621.Remacle J, Bertheau Y. New technology in food sciences facing the multiplicity of new

released GMO (GMOchips); 2001.Remund K, Dixon D, Wright D, Holden L. Statistical considerations in seed purity testing

for transgenic traits. Seed Sci Res 2001;11(2):10119.Rho JK, Lee T,Jung SI,Kim TS, Park YH, Kim YM. Qualitative andquantitative PCR methods

for detection of three lines of genetically modied potatoes. J Agric Food Chem2004;52(11):326974.

Rodriguez-Lazaro D, Lombard B, Smith H, Rzezutka A, D Agostino M, Helmuth R, et al.Trends in analytical methodology in food safety and quality: monitoring microorgan-ismsand genetically modied organisms. TrendsFood Sci Technol 2007;18(6):30619.

Rodriguez-Nogales JM, Cifuentes A, Garcia MC, Marina ML. Estimation of the percentageof transgenic Bt maize in maize our mixtures using perfusion and monolithicreversed-phase high-performance liquid chromatography and chemometric tools.Food Chem 2008;111(2):4839.

RnningSB, VatilingomM, BerdalKG, Holst-JensenA. Event specic real-time quantitative

PCR for genetically modied Bt11 maize (Zea mays). Eur Food Res Technol 2003;216:34754.

Roth L, Zagon J, Laube I, Holst-Jensen A, Broll H. Generation of reference material by theuse of multiple displacement amplication (MDA) for the detection of geneticallymodied organisms (GMOs). Food Anal Methods 2008;1(3):1819.

Rudi K,Rud I,HolckA. A novelmultiplexquantitativeDNA arraybasedPCR (MQDA-PCR) forquanticationof transgenicmaize in foodand feed.Nucleic AcidsRes 2003;31(11):e62.

Russel A, Sparrow R. The case for regulating intragenic GMOs. J Agric Environ Ethics2008;21(2):15381.

Saji H, NakajimaN, Aono M, Tamoki M,Kubo A, WakiyamaS, et al.Monitoring theescapeoftransgenic oilseed rape around Japanese ports and roadsides. Environ Biosafety Res2005;4:21722.

SalviS, D Orso F,Morelli G.Detectionandquanticationof genetically modied organismsusing very short, locked nucleic acid TaqMan probes. J Agric Food Chem 2008;56(12):43207.

Schauzu M. The concept of substantial equivalence in safety assessment of foods derivedfrom genetically modied organisms. AgBiotechNet 2 ABN 044; 2000. p. 14.

Shanahan D, Stokes P, Burns M. A novel method for the detection of Roundup Readysoya using liquid chromatography-electrospray ionisation mass spectrometry.Eur Food Res Technol 2006;225(34):48391.

Shim YY, Shin WS, Moon GS, Kim KH. Quantitative analysis of phosphinothricin-N-acetyltransferase in genetically modied herbicide tolerant pepper by an enzyme-linked immunosorbent assay. J Microbio Biotechnol 2007;17(4):6814.

ShindoY, KuribaraH, MatsuokaT, Futo S,SawadaC, Shono J,et al.Validation of real-timePCR analyses for line-specic quantitation of genetically modied maize andsoybean using new reference molecules. J AOAC Int 2002;85(5):111926.

Singh CK, Ojha A, Kachru DN. Detection and characterization of cry1Ac transgeneconstruct in Bt cotton: Multiplex polymerase chain reaction approach. J AOAC Int2007;90(6):151725.

Singh CK, Ojha A, Bhatanagar RK, Kachru DN. Detection and characterization ofrecombinant DNAexpressing vip3A-type insecticidal gene in GMOs standard single,multiplex and construct-specic PCRassays. AnalBioanal Chem2008;390(1):37787.

Song HS, Kim JH, Kim DH, Kim HY. Qualitative and quantitative analysis of geneticallymodied pepper. J Microbio Biotechnol 2007;17(2):33541.

Stobiecka M, Ciesla JM, Janowska B, Tudek B, Radecka H. Piezoelectric sensor fordetermination of genetically modied soybean roundup ready((R)) in samples notamplied by PCR. Sensors 2007;7(8):146279.

Sun W, Zhong J, Zhang B, Jiao K. Application of cadmium sulde nanoparticles asoligonucleotide labels for the electrochemical detection of NOS terminator genesequences. Anal Bioanal Chem 2007;389(78):217984.

Sun W, Zhong JH, Qin P, Jiao K. Electrochemical biosensor for the detection of cauli-ower mosaic virus 35 S gene sequences using lead sulde nanoparticles asoligonucleotide labels. Analy Biochem 2008;377(2):1159.

TaniH, Noda N, Yamada K,KurataS, TsunedaS, Hirata A,et al.Quantication of geneticallymodied soybean by quenching probe polymerase chain reaction. J Agric Food Chem2005;53(7):253540.

Taverniers I, Van Bockstaele E, De Loose M. Cloned plasmid DNA fragments as calibratorsfor controlling GMOs: different real-time duplex quantitative PCR methods. AnalBioanal Chem 2004;378(5):1198207.

Taverniers I, Windels P, Van Bockstaele E, De Loose M. Use of cloned DNA fragments forevent-specic quantication of genetically modied organisms in pure and mixedfood products. Eur Food Res Technol 2001;213(6):41724.

Taverniers I, WindelsP, VaitilingomM, Milcamps A, VanBockstaele E,Van denEede G, etal.Event-specic plasmid standards and real-time PCR methods for transgenic Bt11,Bt176, and GA21 maize and transgenic GT73 canola. J Agric Food Chem 2005;53(8):304152.

TaverniersI, Papazova N, BertheauY, De Loose M, Holst-Jensen A. Genestacking in transgenicplants: towards compliance between denitions, terminology, and detection within theEU regulatory framework. Environ Biosafety Res 2008;7(4):197218.

Tengs T,Kristoffersen AB, Berdal KG,Thorstensen T,ButenkoM, Nesvold H, et al. Microarray-based method for detection of unknown genetic modications. BMC Biotechnol2007;7:91.

Terry C, Harris N. Event-specic detection of Roundup Ready Soya using two differentreal time PCR detection chemistries. Eur Food Res Technol 2001;213(6):42531.

TheunsI, Windels P, De Buck S, Depicker A, Van Bockstaele E, De Loose M. Identication andcharacterization of T-DNA inserts via T-DNAngerprinting. Euphytica2002;123: 7584.

Trapmann S, Catalani P, Conneele P, Corbisier P, Gancberg D, Hannes E, et al. The

certication of reference materials of dry-mixed soya powder with different massfractions of Roundup Ready Soya IRMM-410S. Report EUR 20273 EN; 2002a.Trapmann S, Schimmel H, Kramer G, Van den Eede G, Pauwels J. Production of certied

reference materials for the detection of genetically modied organisms. J AOAC Int2002b;85(3):7759.

U.S. Department of Agriculture, O.o.I.G.S.R. Audit report. United States Department ofAgriculture controlsover importation of transgenicplantsand animals;2008.p. 126.50601-17-Te.

United States Government Accountability Ofce. Genetically engineered crops. Agenciesare proposing changes to improve oversight, but could take additional steps toenhance coordination and monitoring. United States Government AccountabilityOfce; 2008. p. 1110. GAO-09-60.

Vatilingom M, Pijnenburg H, Gendre F, Brignon P. Real-time quantitative PCR detectionof genetically modied Maximizer maize and Roundup Ready soybean in somerepresentative foods. J Agric Food Chem 1999;47:52616.

Van den Bulcke M, De Schrijver A, De Bernardi D, Devos Y, MbongoMbella G, Casi AL, etal. Detection of genetically modied plant products by protein strip testing: anevaluation of real-life samples. Eur Food Res Technol 2007;225(1):4957.

van Duijn G, van Biert R, Bleeker-Marcelis H, van Boeijen I, Adan A, Jhakrie S, et al.

Detection of genetically modied organisms in foods by protein- and DNA-basedtechniques: bridging the methods. J AOAC Int 2002;85(3):78791.

Vermij P. Liberty Link rice raises specter of tightened regulations. Nat Biotechnol 2006;24(11):13012.

Vollenhofer S, Burg K, Schmidt J, Kroath H. Genetically modied organisms in foodsscreening and specic detection by polymerase chain reaction. J Agric Food Chem1999;47:503843.

Volpe G, Ammid NH, Moscone D, Occhigrossi L, Palleschi G. Development of an immu-nomagneticelectrochemical sensor for detection of BT-CRY1AB/CRY1AC proteins ingenetically modied corn samples. Anal Lett 2006;39(8):1599609.

Waiblinger H, Boernsen B, Pietsch K. GMO routine analysis screening table fordetection of genetically modied plants in food and feed. Dtsch Lebensm Rundsch2008;104(6):2614.

Wall EM, Lawrence TS, Green MJ,Rott ME. Detection and identication of transgenicvirusresistant papaya and squash by multiplex PCR. Eur Food Res Technol 2004;219(1):906.

Wang WY, Fang TJ.Developmentof multiplex andquantitative PCRassayto detectgeneticallymodied roundup ready soybean in foods. J Food Drug Anal 2005;13(2):1328.

WatanabeT, KuribaraH, Mishima T,Kikuchi H, KuboM, Kodama T,et al. New qualitativedetection methods of genetically modied potatoes. Biol Pharm Bull 2004;27(9):13339.

Watanabe T, Tokishita S, Spiegelhalter F, Furui S, Kitta K, Hino A, et al. Development andevaluation of event-specic qualitative PCR methods for genetically modied Bt10maize. J Agric Food Chem 2007;55(4):12749.

Weeks J, Ye J, Rommens C. Development of an in planta method for transformation ofalfalfa (Medicago sativa). Transgenic Res 2008;17(4):58797.

Weighardt F, Barbati C, Paoletti C, Querci M, Kay S, de Beuckeleer M, et al. Real-timepolymerase chain reaction-based approach for quantication of the pat gene in theT25 Zea mays event. J AOAC Int 2004;87(6):134255.

Windels P, Taverniers I, Depicker A, Van Bockstaele E, De Loose M. Characterisation ofthe Roundup Ready soybean insert. Eur Food Res Technol 2001;213(2):10712.

Windels P, Bertrand S, Depicker A, Moens W, Bockstaele E, Loose M. Qualitative andeventspecic PCR real-time detection methods for StarLink maize. Eur Food ResTechnol 2003;216(3):25963.

Wu YH, Wu G, Xiao L, Lu CM. Event-specic qualitative and quantitative PCR detectionmethodsforTransgenicrapeseedhybridsMS1 RF1andMS1 RF2.J AgricFood Chem2007;55(21):83809.

Wu G, Wu YH, Xiao L, Lu CM. Event-specic qualitative and quantitative polymerasechain reaction methods for detection of genetically modied rapeseed Ms8XRf3based on the right border junctions. J AOAC Int 2008;91(1):14351.

Wu G, Wu Y, Xiao L, Lu C. Event-specic qualitative and quantitative PCR detection ofgenetically modied rapeseed Topas 19/2. Food Chem 2009;112(1):2328.

Xu GY, Jiao K, Fan JS, Sun W. Electrochemical detection of specic gene related toCaMV35S using methylene blue and ethylenediamine-modied glassy carbonelectrode. Acta Chim Slov 2006a;53(4):48691.

Xu J, Miao HZ, Wu HF, Huang WS, Tang R, Qiu MY, et al. Screening genetically modi edorganisms using multiplex-PCR coupled with oligonucleotide microarray. BiosensBioelectron 2006b;22(1):717.

Xu J, Zhu S, Miao H, Huang W, Fu Y, Li Y. Event-specic detection of seven geneticallymodied soybean and maizes using multiplex-PCR coupled with oligonucleotidemicroarray. J Agric Food Chem 2007;55(14):55759.

Yamaguchi A, Shimizu K, Mishima T, Aoki N, Hattori H, Sato H, et al. Detection methodof genetically modied papaya using duplex PCR. J Food Hyg Soc Jpn 2006;47(4):14650.

YangLT, Pan AH, ZhangKW,Guo JC, YinCS, Chen JX,et al. Identication andquanticati

2 GMO Holst Jensen

Documents

Transcript of 2 GMO Holst Jensen