5.3. Molecular methods for detection and quantification of GMMs

5.3.1. GMM detection

The first Genetically Modified Organisms (GMO) to be used for health or industrial purposes were modifies strains Escherichia coli and Saccharomyces cerevisiae producing insulin. Since this period numerous recombinant microorganisms have been implemented. They are used in industrial bioreactors under confinement conditions and such processes do not generally require carrying out a GMO detection technology.

The outcome of such a specific need appeared with the introduction in the mid-nineties of genetically modified (GM) animals and plants in the natural environment.

GM crops mainly have opened an important debate, particularly in Western Europe, for safety assessment of food and for potential modification of the environment. Food warranted free from GMO has been proposed to reassure the consumers. However, not only raw materials can be used for food preparation but also numerous ingredients from plant origin, which can be present in small amounts in the final product. It is for example the case with lecithin extracted from Soya beans. Lecithin is used as an additive in a lot of industrial recipes in chocolates of chocolate flavored preparations. The target to be analyzed is not the purified lecithin itself (which is identical to the lecithin from the non-GM Soya beans) but some modified DNA sequences or the coded by them recombinant protein(s). The latter can be only presented as traces, thus very sensitive methods for their detection are necessary.

5.3.2. Types of detection methods

Detection of GMO or its derivative can be performed through detecting of the molecule - primary target (the DNA sequence itself and eventually - RNA), specifically connected with the genetic modification or its product (the recombinant protein that can be produced in relation with the genetic change. The greater part of the methods available concern DNA detection, and just few techniques are applied in RNA and protein detection. The reasons for this fact are as follows:

• DNA can be amplified and purified rapidly and efficiently using PCR. Multiplication of RNA and proteins is more complicated and time consuming process.
• DNA is a stabile molecule while RNA is not stabile at all. The protein is easily subjected to temperature denaturation during food processing, thus its stability depends on various external factors.
• If the modifying element is a nuclear DNA there is a linear dependence of its quantity and the amount of the GMO. However such correlation is virtually not observed between the quantity of the GMO and RNA/Protein.
• Since the genetic modification is done at DNA level, it is reasonable to detect this alternation at the same level.

Nowadays all commercialized GMO possesses foreign nuclear DNA.

5.3.3. Protein based methods

The basis of these methods is immunological and lays in the specific binding of the type “antigen – antibody” and classical ELISA assay. The antigen – antibody reaction recognizes the foreign molecule, binds to it and the bound complex thus obtained, is detected usually through a chromogenic reaction. Since the antibody, needed to detect the antigen, can not be developed without access to the purified antibody itself, the latter can be either artificially synthesized in the amino acid sequence is known, or purified from the GMO studied.

Generally the product of a transgene is a small polypeptide or a protein, which can be expressed under a strong constitutive promoter in any tissue and virtually during any time of the life cycle of the plant. In such a way the protein is in enough quantity to constitute an analytic target. However, in more recent GM plants the desired protein is only produced under the control of an inducible promoter (e.g. under stress conditions, during defined period of life-cycle, etc.). In these situation the detection and analysis of the presence of the recombinant protein is often not adequate.

5.3.4. RNA based methods

In these methods a specific binding between the RNA molecule and a primer (RNA or DNA synthetic oligonucleotide) is performed. The primer, complement to the start of the RNA molecule, annealed with it resulting in a double strand heteroduplex similar to DNA. Using reverse transcriptase a DNA molecule is synthesized de novo, which can be further amplified by PCR and detected. A disadvantage of this method is the fact that the specific primers can not be designed without knowledge about the RNA composition to be detected.

5.3.5. DNA based methods – PCR application

DNA-based methods primary rely on multiplication of a specific DNA piece by PCR technique [14]. For visualization of the amplification products gel electrophoresis is routinely used. It may be solely performed or coupled with restriction endonuclease digestion (RFLP-PCR). A more sophisticated variant of the basic PCR protocol involves determination of the Tm profile by means of a dye intercalating double stranded DNA and emitting fluorescent light. With increasing the temperature the two strands of DNA begin to separate and correspondingly – the light emission, which can be measured, decreases. Tm is a specific characteristic of a DNA sequence rather than DNA length. At last, but not leased an alternative is to use probes and perform hybridization with DNA or RNA. If appropriately designed, a probe can discriminate between the native and any foreign sequence. Labeling the probe with radioactive or non-radioactive compounds facilitates the detection of the present molecule. For GMO analysis gel electrophoresis and hybridization techniques are currently the most commonly exploited techniques.

Screening of food samples for the presence of GMOs by use of basic PCR protocol comprises the following procedure;

• extract of DNA from the sample and standards of unknown GMO content;
• assembly of several PCR with specific primers (usually for well known regulatory sequences, as the viral 35SCaMV or Tnos promoters);
• visualization of the DNA fragments on an agarose gel electrophoresis;
• analysis and semi-quantitative assay using image analysis software.

With multiplex PCR-based methods several DNA sequences can be screened for and detected in a single reaction. However, the development of a multiplex assay requires careful testing and approving. The pool of amplification fragments needs to be further analyzed to distinguish between the various amplicons. This can be done with the aid of specific hybridization probes by gel electrophoresis and comparison of the fragments size or using specifically labeled primers.

A great advantage of this technique is the fact that fewer reactions are needed to test a sample for presence of GMO-derived DNA. Additionally if it is necessary to further perform quantification assays it will be good if you know which GMO to quantify since the procedure is relatively expensive. The identification of a certain GMO is important also in the context of our knowledge about the approved and aunapproved GMOs.

Another approach is to apply PCR-based quantification methods. PCR-based quantification can be performed both during the amplification process (the real-time PCR) and at its end (end-point PCR).

The end product analyses are commonly based on comparison of the mount of amplified DNA of two DNA targets: the one to be quantified and a competitor (in known small quantity) added to the amplification mixture before the PCR and co-amplified with the target to be quantified. This process is also called competitive quantitative PCR. It is based on the presumption that if both target DNA and competitive DNA yield the same amount of amplification product, the starting amount of DNA is also assumed to be the same.

In real-time PCR analysis the amount of a product synthesized during PCR is estimated directly by measurement of the fluorescence in a PCR. There are commercially available hybridization probes emitting fluorescence corresponding to the amount of the synthesized DNA. The amount of the synthesized product can be also estimated by the emitting of intercalated fluorescent dye but here it is not possible to distinguish between specific and not-specific products. The advantage of this method is that not only the quantity of the formed product can be followed in dynamics but also the defined number of cycles, which are needed to produce a certain amount of PCR product, can be determined.

The real-time PCR requires more sophisticated and expensive equipment; it is faster than competitive PCR and more specific.

5.3.6. The detection process

The detection process comprises a procedure consisting of the following individual steps [9]:

1. Sampling. The sampling strategy involves complex statistics to produce reliable estimate of the quantity of GMO or their derivatives.
2. Homogenizing. This step includes homogenization of the sample.
3. Isolation/purification. This step concerns isolation and purification of a DNA, RNA or protein. The most critical factors at this step ate the quantity (concentration), purity and quality of the macromolecule to be tested. Here again statistics are involved.
4. Present/absent analysis. At this step analyses for determination of presence or not of GNO or its derivative are performed. As mentioned above a range of alternative methods is available, each offering different ability to discriminate between derivatives of different GMOs and different reliability concerning false (+) and (-) results. Another considerations that should be taken into account when choosing a detection method are: the probability the molecule to be detected to be entirely degraded, i.e. no longer detectable; the probability during the food processing the molecule, which the detection method is designed to detect, have been removed; the probability the analysis to be performed on a mixture of GMOs and thus the identification process appeared to be more complicated and time consuming.
   1. For identification of the detected molecule usually any (+) result have to be verified in order to omit false (+) reactions and confirm the identity of the found molecule.
   2. The quantitative estimation of the modified material is a sample is performed at this step. Here again statistics are important since quantification always requires standards.
5. Interpretation of the analysis results.

The limits of the detection and quantification can be categorized in three groups:

1. Absolute limits – the lowest number of copies that must present at the beginning of the first cycle to obtain probability of 95 % correct detection;
2. Relative limits – the lowest relative percentage of genetically modified materials that can be detected;
3. Practical limits –the limits applicable to a defined sample.

The specificity of the currently available DNA based methods can be categorized into four groups:

1. Screening methods detecting wide range of GMO without identifying them;
2. Screening methods for a certain type of genetic modification;
3. Construct specific methods sometimes used for identification of GMOs;
4. Transformation specific methods used for identification of GMOs (still under laboratory development; not commercialized).