DIPLOME EN SCIENCES NATURELLES DE L'ENVIRONNEMENT - Universités de Genève et de Lausanne
Mira DAUTI, 2002.
Development of molecular methods for detection and quantification of nanoflagellates in polluted aquifers.
The subject of this study was the phylogenetic analysis of several nanoflagellates,
isolated from groundwater aquifers samples, as well as the development of detection
methods i.e. whole-cell hybridisation. The SSU ribosomal RNA - based approaches
The overall scheme of methodology involved (i) cloning and complete sequencing of SSU rDNA genes obtained by PCR - amplification of extracted nucleic acids from each isolated nanoflagellate, (ii) comparative sequence analysis including SSU rDNA sequences from publicly accessible databases such as GenBank and RDP, (iii) design of group- and species-specific probes, and (iv) their application in whole-cell hybridisation technique.
The SSU rDNA genes of 5 isolated nanoflagellates were sequenced. Three types of isolated nanoflagellates resulted to be phylogenetically related to Heteromita globosa, one type was closely related to two Poterioochromonas species (P. stipitata and P. malhamensis). The SSIJ rDNA gene of the last isolated type could not be amplified and sequenced.
Several group of isolate specific probes were designed to target variable regions of the SSU rRNA of isolated nanoflagellates. But only those targeting the regions V2 and V9 hybridised with the fixed cell of- these flagellates.
The restricted-fragment-length polymorphism (RFLP) method was used to distinguish the flagellates type 1, 2 and 6 from each-other and from H. globosa.
aquifer nanoflagellates, SSU rRNA, phylogenetic affiliation, oligonucleotide probes, whole-cell hybridisation, fingerprinting.