Handbook of Meat Processing

Assessment of Genetically Modifi ed Organisms (GMO) in Meat Products by PCR 509

same species, and preferably, must be present

in the genome in a single copy. Table 29.3

summarizes the endogenous reference control

genes currently available.

Analysis of Transgenes and

Flanking Regions

Different strategies based on PCR amplifi ca-

tion of the introduced transgene have been

defi ned (Holst - Jensen et al. 2003 ): trait -

specifi c methods, where transgene - specifi c

sequence are used for amplifi cation

(Va ï tilingom et al. 1999 ; Zeitler et al. 2002 );

construct or plasmid - specifi c methods, with

the amplifi cation of a fragment containing

two introduced regions from different origins

(Kuribara et al. 2002 ; Hernandez et al.

2004a ); and event - specifi c methods, which

amplify the border region between the intro-

duced sequence and the plant genomic DNA

(e.g., for Roundup Ready soybean: Berdal

and Holst - Jensen 2001 ; Taverniers et al.

2001 ; Terry and Harris 2001 ), for transgenic

maize lines, such as MON810 (Holck et al.

2002 ; Hernandez et al. 2003a ), CBH - 351

(Windels et al. 2003 ), Bt11 (Zimmermann et

al. 2000 ), and NK603 (Nielsen et al. 2004 ).

Table 29.1 summarizes the published GMO

analytical systems based on PCR.

such as pUC19 ) as targets of screening meth-
odologies. Table 29.2 summarizes the avail-
able screening methods published for GMO
analysis.
In addition, virus - infected plants or
samples contaminated with plant material
carrying the virus could lead to false positive
results. To overcome this problem, PCR
systems are available that specifi cally detect
the virus coat protein of all CaMV isolates,
allowing the differentiation of infected plants
and positive GMO samples (Cankar et al.
2005 ).

Identifi cation of Endogenous

Reference Genes

In a food sample, only a certain percentage
may contain the GM ingredient. In order to
identify the total amount of DNA corre-
sponding to that particular GMO, endoge-
nous reference controls that defi ne 100% of
each ingredient must be used. These are
based on genes present in both transgenic and
nontransgenic varieties of the same plant
species. They must not present genetic modi-
fi cations (a SNP was detected in ADH1 gene
affecting quantifi cation; Broothaerts et al.
2008 ), and must show species - specifi city,
stability among different varieties of the

Table 29.2. PCR screening methods for GMO analysis. c PCR : conventional PCR ; RT i - PCR : real - time
PCR Modifi ed from Hernandez et al. (2005)

Target sequence Technique Reference

T - nos cPCR Depicker et al., 1982

npt II cPCR Beck et al., 1982

T - ocs cPCR DeGreeve et al., 1983

pat cPCR T ö pfer et al., 1987

T - CaMV cPCR T ö pfer et al., 1987

npt II, P - 35S, T - nos cPCR Pietsch et al., 1997

P - 35S, T - nos cPCR Brodman et al., 1997

P - 35S, T - nos cPCR Lipp et al., 1999

P - 35S, T - nos, npt II, lec and ivr cPCR Vollenhofer et al., 1999

P - 35S, T - nos qc - PCR Hardegger et al., 1999

P - 35S, T - 35S, P - nos , T - nos,

P - FMV35S, npt II

Multiplex - PCR coupled with

oligonucleotide microarray

Xu et al., 2006

P - 35S cPCR (validation) Weighardt et al., 2004

P - 35S, zeine RTi - PCR duplex H ö hne et al., 2002

bar RTi - PCR Lipp et al., 2001