The identification is based primarily on the analysis of one or two regions of ribosomal DNA: sequencing the D1 / D2 domains of the 28S rRNA gene and/or the ITS region (ITS1 and ITS2 intergenic spaces and 5.8S rRNA gene). The strain can be assigned to a particular species by comparison with sequences available in public databases when the percentage of homology of their sequences is greater than or equal to 99% (Kurtzman and Robnett, 1998; Kurtzman, 2014).
Additionally, sequencing of other genes such as actin, β-Tubulin and /or translation elongation factor 1α, can provide a higher level of resolution in certain groups of microorganisms. The analysis of these sequences enables us to establish phylogenetic relationships and improve identification at species level of those species where ribosomal DNA regions lack resolution. The sequenced genes depend on the taxon to which the isolate belongs.
The identification of filamentous fungi also includes a morphological study to confirm the results obtained in the molecular identification and, in certain cases, to differentiate between species.
A report is provided with data analysis at the molecular level.
In the event that the result is not sufficiently discriminatory to ascertain identification at species level, the CECT staff contact the customer to inform them of the results obtained and the possibility of improving identification through other techniques .
Necessary material
The fundamental requirement for identification is a pure culture of the microorganism
Methodology
The primers used to amplify different ribosomal regions are:
- ITS region (ITS1 and ITS2 including the 5.8S rDNA gene): ITS1-ITS5 / ITS4 (White et al., 1990)
- Domanes D1 / D2 of the 5 'end of 28S rRNA gene: nl1 / NL4 (O'Donnell et al., 1998)
- rRNA gen 28S (1,4Kb): LROR (Cuvette et al., 1991) and LR5 (Vilgalys & Hester, 1990). Internal primers: LR3R / LR3 (Vilgalys & Hester (1990)
The PCR products are checked by electrophoresis on agarose gel 1% (w/v) in Tris-Borate-EDTA buffer at 135 V for 25 minutes.
PCR fragments are sequenced on an ABI 3730xl sequencer (Applied Biosystems) using the same PCR primers at a concentration of 5 nM.
Blast analysis of the sequences obtained against different databases (NCBI or MycoBank, both open access webs) is performed, and our report reflects the overlapping fragment extension, the percentage of similarity and name of the microorganism with a greatest degree of sequence identity.
Bibliography
Cubeta, M.A., E. Echandi, T. Abernethy & R. Vilgalys (1991). Characterization of anastomosis groups of binucleate Rhizoctonia species using restriction analysis of an amplified ribosomal RNA gene. Phytopathology 81:1395-400.
Kurtzman, C.P., & Robnett, C.J. (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek, 73:331–371.
Kurtzman CP (2014). Use of gene sequence analyses and genome comparisons for yeast systematics. International Journal of Systematic and Evolutionary Microbiology, 64:325–332.
O’Donnell, K., Cigelnik, E. & Nirenberg, H. I. (1998). Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia, 90:465–493.
Vilgalys, R. & M. Hester (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172:4238-4246.
White, T. J., T. Bruns, S. Lee, and J. Taylor (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, San Diego, Calif.
