Screening

For the expression of newly integrated genes, GM plant developers use a limited number of regulatory elements (promoters and terminators). Since these elements

Gmo Testing
Fig. 7.2 Procedure for GMO testing of food, feed and seeds from a European perspective. A stepwise approach consisting of GMO screening, identification and quantification is commonly applied for testing food, feed and seed products for compliance with European authorisation and labelling regulations

have been frequently used they are ideal candidates for the screening of a large number of samples and are useful to assess whether or not a sample under investigation is likely to contain GM-derived material (Fig. 7.2). To identify these elements Bruderer and Leitner (2003) systematically surveyed which genetic components have been introduced into GM crops at the worldwide level. Correspondingly, the widely applied screening methods target the constitutive 35S promoter (P-35S) sequence from cauliflower mosaic virus (CaMV) or derivatives of this promoter and the terminator sequence isolated from the nopaline synthase (nos) gene of Agrobacterium tumefaciens which are found in 43 events (P-35S) and in 37 events (T-nos), respectively (Bruderer and Leitner 2003). The survey identified also a few genes with significant numbers of application in GM plants (see Table 7.2). Herbicide-tolerance genes like the cp4epsps gene derived from A. tumefaciens sp. strain CP4, the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus or from S. viridochromogenes ( pat) have been identified to be reasonable targets for screening (Zeitler et al 2002; Waiblinger et al. 2005).

Table 7.2 Genetic elements and constructs present in selected GM crops (Bruderer and Leitner 2003; Waiblinger et al. 2008)

Genetic

GM crop (event name)

element/

Canola

Cotton

Maize

Potato

Rice

Soybean

Sugar beet

construct

P-35S

23-198

MONI 445

676, 678, 680

ATBT04-X (NewLeaf)

LL62

356043

GTSB77

23-18-27 (Laurical)

MONI 69 8

DAS-59122-7

BT X (NewLeaf)

LL06

A2704-12

T120-7

GS40/90

MON15985

B16 (DLL26)

SPBT02-5

LL601

A2704-21

Topas 19/2

31807

Btll

SPT02-7

A5547-127

OXY235

31808

BtlO

A5547-35

Liberator L62

MON531

Btl76

G94-1

T45

757

CBH-351

G94-19

1076

DBT418

G-168

BXN

LY038

GTS40-3-2

MON80100

GU262

MON809

W62

MON8IO

W98

MON832

MON863

MON88017

MON89034

MS3, MS6

NK603

T14, T25

TCI 507

T-nos

MSI

MONI 445

3272

ATBT04-X (NewLeaf)

Bt63

G94-1

RF1

MONI 69 8

Btll

BT X (NewLeaf)

G94-19

RF2

MON15985

CBH-351

RBMT15-X

G-168

MSlxRFl

MON531

GA21

(NewLeaf)

GTS40-3-2

MSlxRF2

757

LY038

RBMT21-X

W62

MS 8

1076

MIR604

(NewLeaf)

W98

RF3

MON80100

RBMT22-X

MS8xRF3

MON802

(NewLeaf)

OXY235

MON809

SPBT02-5, SPT02-7

P35S-pat

GT200 GT73

MSI RF1

MSlxRFl MSlxRF2 MS 8

MS8xRF3

PHY23

PHY36

GS40/90 Topas 19/2 Liberator L62 T45

MONI 445 MONI 69 8

MON832

MON863

MON88017

MON89034

MS3, MS6

NK603

MON8OIOO

MON802

MON809

MON832

MON88017

NK603

B16 (DLL26)

Btl76

CBH-351

DAS-06275-8

DBT418

MS3, MS6

676,678,680

DAS-59122

DAS-59132

Btll

T14, T25

TC1507

RBMT22-X (NewLeaf)

MON89788 GTSB77 MON87754-1 H7-1

LL62 W62 LL06 W98 LL601

A2704-12 T120-7

A2704-21 T252

A5547-35 A5547-127 GU262

The 8 endotoxin (cry) genes from Bacillus thuringiensis (see Chap. 10) belong to the most frequently used genes in transgenic crops. However, screening methods targeting the different cry genes have not been established, because theses genes and gene variants are target-organism specific, often synthetic or modified and in some cases truncated or fused, thus making this gene group less suitable for screening purposes.

If for example canola seed samples (canola see Chap. 21) have to be screened for the presence of GM events it is not advisable to use the P-35S specific method, since many GM canola events remain undetected (Table 7.2) and CaMV can infect rapeseed, thus increasing the chances of false-positive results. A screening concept for canola seeds proposed by the German official control laboratories therefore applies a combination of four different construct-specific PCR tests, allowing the detection of 13 known GM canola events (LAG 2006). As described in this concept, the combination of four PCR tests (P35S-pat, pFMV-epsps, pSSUAra-bar, P35S-nptII) covers 13 events and, if one test is positive, further analyses for identification of the GM event have to be performed.

Recently, also real-time PCR arrays based on multi-target analytical systems were developed to serve as less laborious analytical tools for the screening of unauthorised GM crops in the EU and Japan (Querci et al. 2008; Mano et al. 2009). The formats are 96-well or 384-well PCR plates prepared with primers and probes specific for the simultaneous detection of as many GM elements, constructs and events as possible.

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