5.2. Molecular methods for control of genetically modified foods

The taxonomic status of the host microorganism is considered to be a feature of great importance regarding safety assessment. For this reason, the microorganisms used for genetic manipulations should be well examined taxonomically using proper methodology. It should be adequately characterized from scientific, manufacturing and safety perspective. At present, the most exact tool for proper characterization of the taxonomic status of microorganisms are DNA/DNA hybridization technique and 16S rRNA sequence determination. These methods give crucial information about taxonomical status of the microorganisms under investigation. At present standard physiological/biochemical methods for phenotypic characterization are on the market and are widely used. The important feature for the strain characterization is also information about its pathogenic properties.

After application of genetic modification procedure the obtained GMM strains should sustain the safe properties of the host microorganisms. The new strain should be characterized by the same methods and accuracy, including phenotypic and genotypic characteristics in order to assess its safety. This precise comparison between the host and GMM could be done using existing molecular techniques: restriction analysis, random amplified polymorphic DNA analysis (RAPD-PCR), amplified fragment length polymorphism (AFLP), protein profiling etc. The analysis can be extended also to genome sequencing.

Other important factors, which should be studied in respect to the safety assessment of GMM are: the effect of the genetic modification on the properties of the host microorganism, the stability of the genetic system, the functional properties of the gene construct.

All these characteristics are important during the process of safety assessment of the products obtained by GMMs and their impact on the environment.

The methods for identification of production strains, contaminating strains or pathogens.comprise techniques applied at both genotype and phenotype level. The genotypic methods include tools such as rDNA sequence analysis, DNA base composition and DNA/DNA hybridization.

DNA sequencing, especially rDNA sequence analysis aims at comparative studies of rDNA sequences. This is performed through direct sequencing of parts or nearly the entire 16S or 23S rDNA molecule by PCR using appropriate primers.

DNA base ratio (moles percent G + C) is a classical genotyping method, part of the standard description of bacterial taxa. The range observed is no more than 3 % within a species and no more than 10% within a genus. Among bacteria G + C content varies between 24 and 76 %.

DNA/DNA hybridization is applied for identification virtually to all bacteria, as well as to great variety of yeasts and fungi.

As regards the taxonomic resolution of these methods rDNA sequence analysis and DNA base composition are readily applicable for genus and species identification, while the DNA/DNA hybridization is used only for species characterization. The genotyping methods have useful application for bacteria and yeasts and to some smaller extent to fungi (only rDNA sequence analysis).

The phenotyping molecular methods include cellular fatty acids fingerprinting and total cellular protein electrophoretic patters. These methods are mainly used in bacterial identification and while the former is applicable to genus and species level, the latter is routinely used for species identification.

5.2.1. Strains identification methods

The taxonomic status of the host microorganism is considered to be a feature of great importance regarding safety assessment. For this reason, the microorganisms used for genetic manipulations should be well examined taxonomically using proper methodology. It should be adequately characterized from scientific, manufacturing and safety perspective. At present, the most exact tool for proper characterization of the taxonomic status of microorganisms are DNA/DNA hybridization technique and 16S rRNA sequence determination. These methods give crucial information about taxonomical status of the microorganisms under investigation. At present standard physiological/biochemical methods for phenotypic characterization are on the market and are widely used. The important feature for the strain characterization is also information about its pathogenic properties.

After application of genetic modification procedure the obtained GMM strains should sustain the safe properties of the host microorganisms. The new strain should be characterized by the same methods and accuracy, including phenotypic and genotypic characteristics in order to assess its safety. This precise comparison between the host and GMM could be done using existing molecular techniques: restriction analysis, random amplified polymorphic DNA analysis (RAPD-PCR), amplified fragment length polymorphism (AFLP), protein profiling etc. The analysis can be extended also to genome sequencing.

Other important factors, which should be studied in respect to the safety assessment of GMM are: the effect of the genetic modification on the properties of the host microorganism, the stability of the genetic system, the functional properties of the gene construct.

All these characteristics are important during the process of safety assessment of the products obtained by GMMs and their impact on the environment.

The methods for identification of production strains, contaminating strains or pathogens.comprise techniques applied at both genotype and phenotype level. The genotypic methods include tools such as rDNA sequence analysis, DNA base composition and DNA/DNA hybridization.

DNA sequencing, especially rDNA sequence analysis aims at comparative studies of rDNA sequences. This is performed through direct sequencing of parts or nearly the entire 16S or 23S rDNA molecule by PCR using appropriate primers.

DNA base ratio (moles percent G + C) is a classical genotyping method, part of the standard description of bacterial taxa. The range observed is no more than 3 % within a species and no more than 10% within a genus. Among bacteria G + C content varies between 24 and 76 %.

DNA/DNA hybridization is applied for identification virtually to all bacteria, as well as to great variety of yeasts and fungi.

As regards the taxonomic resolution of these methods rDNA sequence analysis and DNA base composition are readily applicable for genus and species identification, while the DNA/DNA hybridization is used only for species characterization. The genotyping methods have useful application for bacteria and yeasts and to some smaller extent to fungi (only rDNA sequence analysis).

The phenotyping molecular methods include cellular fatty acids fingerprinting and total cellular protein electrophoretic patters. These methods are mainly used in bacterial identification and while the former is applicable to genus and species level, the latter is routinely used for species identification.

5.2.2. Typing methods

Introducing molecular biological techniques a variety of DNA-based typing methods for discrimination of species, as well as isolates of a certain species are designed. The data obtained applying these methods may provide insight in the dissemination and persistence of food spoiling microorganisms or pathogenic ones not only in the foods but also in the environment. Thus, DNA-based typing methods can be used for epidemiological purposes and can help discriminating coincident but independent infections and epidemics caused by a single isolate. This is of special importance since may facilitate the implementation of preventive and hygienic measure.

Genotyping methods are usually categorized depending on technical aspects. According to this the following can be listed:

• DNA sequencing;
• Restriction endonucleases patterns analysis of plasmid and/or genomic DNA (e.g. Restriction Fragments Length Polymorphisms (RFLP), Pulse Filed Gel Electrophoresis (PFGE));
• Probe-based techniques (labeling methods);
• PCR-based techniques (amplification methods such as Random Amplified Polymorphic DNA (RAPD) and Amplified Fragments Length Polymorphisms (AFLP)).

Restriction Fragments Length Polymorphisms (RFLP) explores the natural variability of DNA molecules (chromosomes, plasmids and mitochondrial DNA in eukaryotes) regarding the position and the number of 6- to 8-mer sequence along this molecule. Cutting such DNA with restriction endunucleases results in generation of fragments with different length which can be separated by Agarose Gel Electrophoresis (AGE) and visualized either directly after staining with ethidium bromide (in case of limited number of fragments, e.g. less than 50) or after hybridization with specific labeled probes.

Pulse Filed Gel Electrophoresis (PFGE) is a technique explored in separation of large DNA molecules such as chromosomes. It can be applied also for separation of large DNA fragments obtained after cutting with rare-cutting restriction endunucleases, which generate a limited number of fragments. Being highly polymorphic the chromosomes/large fragments patterns are very useful for strain identification.

Probe-based methods (labeling methods). These methods concern incorporation into or attachment at the end of a nucleic acid fragment a probe. Different variations of the basic method exist depending on variety of factors. As concern the type of the nucleic acid, its size and quantity, 4 methods of labeling can be listed: 3’ and 5’ end labeling, random labeling by nick translation and random prime labeling. In respect to the labeling molecule, i.e. its nature, radioactive and non-radioactive labeling can be explored. In the radioactive labeling radioisotopes are detected by autoradiography while the non-radioactive labeling utilizes fluorescence, chemo-luminescence or enzymatic reactions.

PCR-based techniques (amplification methods). In these methods the in vitro enzymatic amplification of nucleotide sequence is explored for strain identification purposes. Both basic PCR protocol and its modifications are powerful tools for amplification of a DNA sequence of interest, detection and typing of production, contaminating and pathogenic strains. Among the wide diversity of PCR-based methods targeting direct identification of a given organism in a food product the RAPD and AFLP analyses are the most commonly used.

Random Amplified Polymorphic DNA (RAPD) is a random type PCR technique that is based on amplification of a DNA region without previous information about some target sequence. Designing of 10-mer primers at random and applying of low stringency control amplification conditions a set of amplified sequences can be obtained, which in general is individual specific. This last fact contributes to the use of RAPD-PCR as a reliable method to differentiate among microorganisms populations.

Amplified Fragments Length Polymorphisms (AFLP) is a highly sensitive method for detection of polymorphism among individuals applicable for both inter- and intra-species discrimination. This is PCR mediated RFLP of selected DNA fragments from a pool of such. AFLP screens for polymorphism in the length of the amplified fragments through selective amplification of some of them. The method comprises digestion of genomic DNA with 2 restriction endonucleases followed by PCR amplification of the obtained fragments. The restriction fragments are modified in advance to the amplification with adaptors specific for the endonucleases used in the experiment, thus serving as primer binding sites. The primers themselves are designed in a way that bind to the adaptors and allow very specific amplification due to the fact that only fragments fully matching the primers sequence will be amplified.

5.2.3. Strains detection and tracing methods

Here, only genotyping methods are explored, namely all typing techniques listed above with special emphasis on DNA probes, which discrimination power covers detection at genus, species and strain level in bacteria, yeasts and fungi.