Contents - Faperta

Detection and Monitoring of Food and Food Products 471
hybridization phase, the labeled fragments associate with the spotted probes on the basis of
complementary sequences. The larger the number of complementary sequences, the stronger
the bond will be. After the hybridization phase, the free labeled sequences and weakly
bound probes are washed off, and the array can be scanned for fl uorescence intensity of
each spot. Analysis of the resulting patterns and intensity gives an idea of the presence of
genetically modifi ed food. It can also be used for protein-based methods. Promising new
developments in this area are the development of an electroarray system, where the fl uorescently
labeled negative DNA fragments are guided to individual spots that are positively
charged in order to increase the rate of hybridization events (Nanogen, 2003). Systems that
increase the surface where hybridization may occur by using three-dimensional spot structures
such as gel-based chips have also been developed (Miraglia et al., 2004).
RNA-Based Methods
The RNA-based methods rely on specifi c binding between RNA molecule and the
synthetic RNA molecule called a primer. The primer should be complementary to the
nucleotide sequence at the start of the RNA molecule. The binding between the RNA molecule
and the primer is followed by conversion of RNA to a DNA molecule through a
process called reverse transcription. Finally, the DNA can be multiplied by PCR or translated
into over 100 copies of the original RNA molecule, and the procedure repeated by
using each copy as a template using the NASBA (nucleic acid sequence-based amplifi cation)
technique. The specifi c primers needed for the process can be developed without prior
knowledge of the composition of the RNA molecule to be detected (Holst-Jensen, 2006).
Protein-Based Methods
Immunoassay can also be used for detection and quantifi cation of foreign proteins introduced
through genetic transformation of plants. Immunoassay is based on the specifi c
binding between an antigen and an antibody. Thus, the availability of antibodies with the
desired affi nity and specifi city is the most important factor for setting up an immunoassay.
Immunoassays can be used qualitatively or quantitatively over a wide range of concentrations.
Western blot, enzyme-linked immunosorbent assay (ELISA), and lateral fl ow sticks
are typical protein-based methods. The antibodies can be polyclonal (raised in animals) or
monoclonal (produced by cell cultures). Commercially available polyclonal antiserum is
often produced in rabbits, goats, or sheep. Monoclonal antibodies offer some advantages
over polyclonal antibodies because they express uniform affi nity and specifi city against a
single epitope or antigenic determinant and can be produced in vast quantities. Both polyclonal
and monoclonal antibodies may require further purifi cation to enhance sensitivity.
The specifi city of the antibodies must be checked carefully to elucidate any cross-reactivity
with similar substances, which might cause false positive results.
Enzyme-Linked Immunosorbent Assay
In ELISA, the antigen–antibody reaction takes place on a solid phase (microtiter plates).
Antigen and antibody react and produce a stable complex, which can be visualized by the
addition of a second antibody linked to an enzyme. Addition of a substrate for that enzyme
results in color formation, which can be measured photometrically or recognized by the
naked eye. ELISA test kits provide semiquantitative results in a short time with detection
limits less than 0.1%. ELISA has been designed to detect a novel transgene protein or trait.