Immunoassays, PCR```

Methods for the identification of GM organism in food can be divided into 3 categories. In the first category are nucleotide-based amplification methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), fingerprinting techniques (such as RFLP, AFLP, and RAPD), probe hybridization, “self-sustained sequence replication” (3SR), and “Q replicase amplification”. The second category involves protein-based methods including one dimensional SDS gel electrophoresis, two-dimensional SDS gel electrophoresis, Western-blot analysis and ELISA(enzyme-linked immunoabsorbant assay). The third category is based on the detection of enzymatic activities.
Every detection method has its own specificity and limitations. The detection using an enzymatic activity method is not recommended for processed foods, where proteins may be denaturized.



PCR:
The methods based on PCR are not suitable for detection of highly processed foods because DNA fragments in foods could be broken into pieces. Among the 3 categories, PCR is the most popular method used worldwide. The key elements in the PCR process are as follows:
“primers”—small DNA molecules whose sequences correspond to the target sequence.
A heat stable DNA polymerase—typically Taq polymerase, which synthesizes new copies of the target sequence in a manner that is dependent upon the interaction of primers with these target sequences.
A thermocycler—an apparatus that can be programmed to carry the contents of the PCR reaction vessels through multiple, precisely controlled temperature cycles.



Immunoassays:
Immunoassays are ideal techniques for quantitative and qualitative detection of a variety of proteins in complex material when the target analyte is known. An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets Immunoassays utilizing high affinity polyclonal antibodies resulted in high throughput, sensitive and specific in vitro analytical procedures.

(An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]

Near infrared spectroscopy:
NIR spectroscopy relies on the relationship between a sample’s absorbance of incident NIR radiation, and the concentration of absorbing species within the sample. When NIR radiation impinges on a sample, certain molecules with the sample will vibrate depending on their mass, chemical bonding structure, and the wavelength of the incident radiation. This relationship makes vibrational spectra useful for gaining insight into the molecular structure of a compound.

Hybridization
It comprises amplifying transgenes of GMO by biotin-labeled primer sets, hybridizing the amplified products with colored bead-labeled probes, and detecting the hybrids.