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.