3 Recent Advances 57for cultivation and commercialization grows, there
is also an increased risk of transgenic material con-
tamination in nontransgenic food products. One such
well-publicized event took place in October 2000,
when Safeway and Taco Bell recalled corn products
because they were contaminated with small amounts
of genetically engineered corn. For this and other
reasons, the future success and acceptance of GMOs
will depend on mechanisms for containment and
proper detection of transgenic material. Among the
different methods for detecting transgenic materials
that are in use today, real-time quantitative PCR is
the most powerful, accessible, and cost efficient
(Higuchi et al. 1992). The main concern for the
implementation of reliable detection methods is to
determine what type of unique gene sequence
should be amplified during the PCR screening.
Signature sequences such as antibiotic resistance
markers and promoters are the main elements used
today for detection of GMOs, but they are not ideal
since the same signature sequences can be found in
more than one type of GMO. Also, there is an un-
proven concern that these signature sequences, espe-
cially antibiotic resistance markers, may cause
health and environmental problems. To address this
concern, the European Union, which has adopted
stringent regulation on GMOs, banned the use of
antibiotic gene as markers for transformation selec-
tion, by the year 2004. The European Union also
established mandatory labeling of GMO foods with
a 1% threshold level for the presence of transgenic
material, which in turn encouraged more aggressive
research on highly specific, precise, and sensitive
methods for detection and quantification of GMOs
in food products (European Commission 2000).
Researchers for the German company Icon Ge-
netics developed a novel idea for universal identifi-
cation of GMOs (Marillonet et al. 2003). They pro-
posed the creation of a standardized procedure in
which nontranscribed DNA-based technical infor-
mation can be added to the transgene before it is
inserted in the organism’s genome. This artificial
coding would be based on nucleotide triplets, just
like amino acids codons, and each triplet would
encode for one of the 26 Latin alphabetic letters, an
Arabic numeral from 0 to 9, and one space character,
giving a total of 37 characters (Table 3.2) (Maril-
lonet et al. 2003). With these characters, the re-
searchers could insert biologically neutral, nonge-
netic coding sequences that translate into unique
information such as the name of the company, pro-
duction date, place of production, product model,
and serial number. The variable region where the
information is encoded will be cloned between con-
served sequences that contain primer-binding do-
mains. To read the DNA-encoded information, one
only needs to perform PCR and sequence the frag-
ment.
Another PCR-based method for GMO detection
involves the use of unique genomic sequences flank-
ing the transgene. Hernandez et al. (2003), working
with Monsanto’s transgenic maize line MON810,
which contains a gene encoding for the insecticide
CryIA(b) endotoxin, identified a genomic sequence
adjacent to the 3-integration site of the transgenic
plant by using a thermal asymmetric interlaced
(TAIL)-PCR approach. PCR amplification of target
DNA and real-time PCR product quantification are
the two most used techniques for accurate DNA
quantification. Real-time quantitative PCR can be
used with different quantitative tools such as DNA-
binding dyes (Morrison et al. 1998), fluorescent
oligonucleotides (Whitcombe et al. 1999), molecu-
lar beacons (Tyagi and Kramer 1996), fluorescence
resonance energy transfer (FRET) probes (Wittwer
et al. 1997), and TaqMan probes (Heid et al. 1996).
The main advantage of the TaqMan system is that it
is highly specific because it uses three oligonu-
cleotides in the PCR reaction. This detection system
consists of two primers that are responsible for
product amplification, and the TaqMan probe, a flu-
orogenic oligonucleotide that will anneal to the
product. During amplification, Taq polymerase re-
leases a 5fluorescent tag from the annealed Taq-
Man probe, which gives off a quantifiable fluores-
cence light. Higher light intensity translates into a
greater amount of the target gene present in the food
sample.FOODPATHOGENDETECTIONFood poisoning may occur due to contamination of
food by certain toxin-producing bacteria such as
Salmonella, Vibrio, Listeria, and E. coli. The strain
O157:H7 is the deadliest among all E. colistrains; it
produces toxins called Shiga, which are encoded by
two genes, stx1and stx2.Shiga toxins (Stx1 and
Stx2) damage the lining of the large intestine, caus-
ing severe diarrhea and dehydration; and if absorbed
into the bloodstream, the toxins can harm other