Genetic Detection Limited

Identification Methods

 

Techniques Used in the Identification of Micro-organisms

Bacterial pure cultures

Bacterial pure cultures

To identify Bacteria and Archaea, we use 16S rRNA as a specific phylogenetic marker. DNA is extracted from cultures and the 16S rRNA gene sequenced. The identification is then carried out by comparative sequence analysis to the TREE databank, which contains more than 20,000 sequences.

For more specific identification of particular functional microbial groups, we employ PCR primers targeting particular function-specific genes, and apply comparative sequence analysis to extensive databanks of these genes.

The customer receives a complete analysis report containing a phylogenetic tree showing the position of the culture relative to other prokaryotes and a list of the ten most closely related organisms.

Fungal pure cultures

Fungal pure cultures

For fungal identification we sequence the ribosomal 18S rRNA gene. Comparative sequence analysis is performed against the TREE databank. For closer identification, the ITS- (intergenic transcribed spacer region) and the IGS-(intergenic spacer region) are analysed. The morphology and developmental stage of the fungus are also described.

The customer receives an extensive analysis report which contains a phylogenetic tree and a list of the ten most closely related organisms. The report also includes a complete summary of the characterisation of the fungus, highlighting any special or unusual properties determined.

Strain level identification

Strain level identification

The following strain level identification methods are used: RAPD-PCR (Random Amplified Polymorphic DNA); RFLP (Restriction Fragment Length Polymorphism) and T-RFLP (Terminal Restriction Fragment Length Polymorphism)

These methods are used for the quality control of pure cultures. They are also used to determine if two organisms that are identical, based on biochemical and physiological tests, are also phylogenetically identical.

Molecular biological identification of moulds and dust samples

Moulds

Moulds are present everywhere around us. They can cause problems in buildings by eliciting allergic reactions. Moreover, they can destroy wood and paper material leading to damage of buildings, artworks and cultural assets.

If the moulds grow continuously, it becomes necessary immediately to identify and classify them to take adequate counteractive measures. Up to now, the limited classical methods of cultivation and microscopic determination have been applied which can be time-consuming.

Therefore, we use modern molecular biological methods for identification. For this, cultivation of the organism is not necessary. For the identification, a specific part of the genetic material is amplified via PCR and the sequence is determined. With the TREE database, the sequence is then analysed.

This database contains more than 20,000 sequences covering a large spectrum of prokaryotic and eukaryotic organisms. Through the use of specific primers and sensors, a very exact identification of the mould is possible.

Furthermore, we determine the fungal concentration of dust samples occurring in living areas but also in museums after long-lasting exhibitions of artworks.

Quantitative analysis of micro-organisms in mixed cultures

For the quantitative analysis of micro-organisms we recommend molecular biological methods, in particular fluorescence in situ hybridisation (FISH) and quantitative real-time-PCR.

In FISH, oligonucleotide probes labelled with a fluorescent dye are employed. These probes can be designed specifically to target individual species, so that an exact estimate of the population sizes of particular microbial species can be made in a sample containing diverse species. The analysis involves fixation of the sample, hybridisation with the fluorescent probes, and examination by epifluorescence microscopy, all of which are performed in our laboratories.

The customer receives a report which includes the results of the population counts and, on request, photographic documentation of the analysis.

Quantitative real-time-PCR allows a rapid and sensitive quantitative assessment of microbial populations. It is based on the PCR reaction and the examination of the product, this is facilitated by the use of oligonucleotide probes labelled with fluorescent dyes. Like FISH, this method allows the quantification of individual species in a sample containing diverse micro-organisms.

One useful application of this method is quantitative detection of pathogenic organisms in liquid and solid samples. Its primary advantage over cultivation-dependent methods is the speed of analysis. A result is obtainable within 6 hours of sample receipt in the laboratory. The TREE databank also allows specific primer systems to be designed for most micro-organisms.

With this method the customer avoids potentially costly delays between sampling and obtaining results.

Qualitative identification of micro-organisms in mixed cultures

To identify and characterise the micro-organisms present in complex environmental samples in the shortest possible time, we employ molecular biological methods.

We perform polymerase chain reaction (PCR) -based methods on DNA extracted from a sample. The PCR reaction is performed with standard primers, or with primers developed specifically by us to target a desired micro-organisms. Then the PCR products are cloned and DNA from the clones is sequenced.

The DNA sequence data obtained is analysed against the TREE Databank, and, if desired, a phylogenetic tree is constructed showing the taxonomic position of the micro-organisms detected.

The customer receives a final report which includes the sequence data and the phylogenetic tree. When desired, the results are presented in a publication-quality format.

Detection of Mycoplasma

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