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.