Handbook of Meat Processing

Assessment of Genetically Modifi ed Organisms (GMO) in Meat Products by PCR 511

evant issues such as the use of certifi ed mate-

rial and the validation and implementation of

useful methodologies in food - industrial pro-

cesses must be carefully considered. The

Community Reference Laboratory for GM

Food and Feed of the Joint Research Centre

(JRC) of the European Commission has

defi ned minimum performance requirements

for analytical methods of GMO testing

( http://gmo-crl.jrc.ec.europa.eu/doc/Min_

Perf_Requir_Analyt_methods_131008.pdf ).

Use of Certifi ed Reference Materials

The use of certifi ed reference materials

(CRM) is needed in RTi - PCR systems for

assessment of GMO quantifi cation. The ref-

erence materials used are either GM raw

materials (i.e., fl our where DNA must be

purifi ed) or plasmids (that contain the

required targets) (Taverniers et al. 2001,

2004 ; Kuribara et al. 2002 ). Ideally, the refer-

ence material should simulate the sample

under study as much as possible, although

this is not always feasible due to the wide

range of food matrices (Yates 1999 ).

Furthermore, DNA content per mass must be

considered because it fl uctuates among dif-

ferent cultivars of the same species due to the

variation of the DNA content of endosperm,

embryo, and teguments of kernels (Trifa and

Zhang 2004 ). Currently, only high - quality

DNA samples purifi ed from certifi ed raw

materials or plasmids are being used as refer-

ence controls to determine the ratio of trans-

genic DNA to total. It is still a matter of

debate whether genomic DNA extracted

from certifi ed raw materials better qualifi es

as a standard compared with the use of plas-

mids containing the target sequences. While

the genomic certifi ed material better mimics

the target of detection in the real sample, it

is sometimes diffi cult to obtain and is expen-

sive. The use of plasmids as standards has

been recently introduced (Hernandez et al.

2003a ; Taverniers et al. 2004, 2005 ; Toyota

et al. 2006 ), with the advantages of being

Schmittgen 2001 ); recently, for example, a
validated quantitative RTi - PCR method for
MON 810 has been assessed using this
approach (Aguilera et al. 2009 ).
The lowest serial dilution allows the
determination of absolute detection and
quantifi cation limits of the method, while a
correct determination of the practical quanti-
fi cation limits requires a thorough statistical
examination of the sampling procedure when
preparing the serial dilutions, based on bino-
mial distributions and Monte - Carlo simula-
tions (Hernandez et al. 2003a ). Finally,
careful analysis and interpretation of data
from the real - time PCR method have to be
performed properly, with the use of the
correct calculation and interpretation of cor-
relation coeffi cients and regression tech-
niques, especially when traces are analyzed
(Burns et al. 2004 ).

Multiplex PCR

The number of commercialized GMOs is
constantly increasing in the market; there-
fore, routine methodologies for GMO
detection adapted to multiple analyses are
convenient. Multiplex PCR requires several
primers that lead to amplifi cation of unique
DNA regions under a single reaction ( Atlas
and Bej 1994; Elnifro et al. 2000 ; Wittwer et
al. 2001 ). This approach is cheaper and less
labor intensive, saving time and effort.
However, the use of many PCR primers in a
single tube can cause some problems, such
as the increased formation of misprimed PCR
products or “ primer dimers, ” and the ampli-
fi cation discrimination of longer DNA frag-
ments (Higuchi et al. 1992 ; Atlas and Bej
1994).

Application of Current

Methodologies to GMO Analysis

The ultimate purpose of the development of
any method for GMO analysis is its practical
application in food analysis. Therefore, rel-