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Volume 2
     

Micro-arrays for the detection of the abundance and distribution of pathogenic protozoa, flagellated algae and diatoms (MICROPAD)

   
Project

QLK3-2002-01939

Cell factory area

3.2.2

EU Contribution

779,536 Euro

Duration

36 months

Type

RS

Starting date

01 October 2002

Keywords
artificial neural networks
fluorescent labelling
molecule markers
oligonucleotide probes
ABSTRACT

The environment is experiencing rapid and accelerating changes, largely originating from human activity, whether coming from local impacts or from the more dispersed effects of global climate change. Only now are we developing the molecular tools required to adequately document microbial diversity on a routine basis. These tools are arising from the combination of three powerful techniques, namely analytical flow cytometry, artificial neural nets and molecular probes. The latter coupled with micro-array construction provides a novel technique to quantify the abundance and distribution of micro-organisms in a rapid and sensitive method. Phylogenetic micro-arrays will be developed to assess water quality, to monitor biodiversity of organisms difficult to identify by traditional means and to compare with traditional means of assessment.

OBJECTIVES

The major objectives include:

  1. new methodologies to assess biodiversity and dynamics.
  2. bioassays for water quality based on molecular methods.
  3. studying diversity patterns in relation to biotic and abiotic factors.
  4. developing molecular markers that can be used in new strategies for identification and monitoring of micro-organisms.
  5. developing the use of artificial neural networks (ANNs) to display the temporal and spatial relationships among the isolates and the use of ANNs and microarrays to model algal successional models from long-term series data.
DESCRIPTION OF THE WORK

In this project each partner will determine or extract from the public domain a suite of potential rRNA probes that are applicable for the group of target organisms of interest to estimate their presence and abundance through time and space. These probes will then be used to develop microarrays to determine the biodiversity of their target organisms at selected sites in a spatial and temporal pattern. For this purpose the partners will search the database for suitable sequences or determine appropriate sequences in order to develop species-specific oligonucleotide probes from rRNA genes for target specific species, namely diatoms (P1), pathogenic protozoa (P2) and flagellated algae (P3).

  • develop microarrays for the detection of selected protozoan (P2) and algal and diatom species (P1 and P3).
  • take marine or fresh (P1 and P3) and waste water (P2) samples.
  • make RNA/DNA extractions from the environmental samples (P1, P2 and P3).
  • make a PCR reaction to fluorescently label the PCR amplified copies of the rRNA/DNA (P1, P2 and P3).
  • spot and hybridise the fluorescently labelled PCR products onto the microchip with species specific oligonucleotide probes (P1, P2 and P3).
  • based on the relative fluorescent signal of the microarrays, compute in silico the abundance and distribution of target species in the environmental samples and compare the results with those obtained by traditional detection means (P1, P2 and P3).
DELIVERABLES
  • compilation of rRNA sequence data for target diatom, protozoan and algal species.
  • design of probes for target species.
  • specificity tests of all probes for target species.
  • design, production and tests of microarrays for target species.
  • microarrays for the detection of diatoms in fresh water samples, pathogenic protozoa in aquatic environments and flagellated algae in coastal marine ecosystems.
CONSORTIUM
COORDINATOR
 
Claudio O. Gualerzi
Department of Biology MCA
University of Camerino
62032 Camerino (MC), Italy
Tel: +39-0737-403 240
Fax: +39-0737-403 243
claudio.gualerzi@unicam.it
 
PARTNERS
 
Gerard Muyzer
Kluyver Laboratory for Biotechnology
Delft University of Technology
2628 BC Delft, The Netherlands
Tel: +31-15-278 1193
Fax: +31-15-278 2355
G.Muyzer@tnw.tudelft.nl

Linda Medlin
Department of Biological Oceanography
Alfred-Wegener-Institute for Polar and Marine Research
27570 Bremerhaven, Germany
Tel: +49-471-1831 1443
Fax: +49-471-1831 1425
lmedlin@awi-bremerhaven.de