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ASSESSMENT OF A METHODOLOGY FOR THE FAST DESIGN OF FUNGAL DNA PROBES AND PCR TAGS

Objective

One key need of fungal biotechnology and molecular biology is the design of non ambiguous DNA probes and PCR markers. For patenting and quality control, molecular signatures or markers are useful for identifying natural organisms and their recombinant derivatives. For biosafety, similar non ambiguous markers are a prerequisite for regulatory procedures since EEC regulations request identification and detection tools for genetically modified organisms. The subject of this concerted action is the assessment of a methodology for the rapid design of fungal PCR tags, markers and DNA probes. Such methodology can be considered as a model applicable to other taxonomic groups.
The objective of the project is to use polymerase chain reaction (PCR) primer design for fungal molecular typing and taxonomy; identification and ownership certification; environmental and health biosafety; and description of fungal biodiversities. Fungal strains representative of the major genera and species involved in several areas of biotechnology have been selected to be studied under 2 coordinated approaches; taxonomy and culturecollection standardization; and molecular typing (including validation and sampling) and computerization of results. Potential genera, species, strain specific PCR primers and derived deoxyribonulceic acid (DNA) probes will be designed and validated. The specificity of the primers or probes will be defined and controlled on the basis of the ecological niches which belongs to the fungi of interest.

To date a set a species-specific PCR primers for fungi pathogenic to humans has been designed along a background of 32 species involving a set of 90 strains. With the development of the DNA bank and ribosomal DNA sequence database the rate at which PCR primers can be designed for a given species in a ecological niche should increase rapidly. The project is continuing.
Two actions will be organized:

a bibliographic review of the last development in fungal molecular biology, biotechnology will be made with particular attention given to the identification problems in patenting and biosafety.
coordinated scientific research applying the same methodology to a collection of selected fungal species and genera.

Methodology
Principle: The basic and simple idea is to amplify genomic regions of interest by PCR, to sequence and align the sequences, to design PCR tags and identify genera or species specific PCR markers. The validated PCR markers can then, outside the frame of this concerted action, be used for detection and identification in luminescence based or similar methodologies.
Genomic regions of interest:

predetermined regions;
the 18s ribosomal subunit;
the 18s/28S ribosomal spacer region;
the adjacent sequences upstream of the 18S ribosomal subunit.
Undetermined regions.

In this option, random DNA amplification fingerprints are computerized to produce genera/species specific statistical envelope profiles. Differences between envelope profiles identify those DNA sequences of potential use as DNA probes and/or PCR tags.

Coordinated assessment of the methodology in the case of fungal species.

Fungal strains representative of the major genera and species involved in several areas of biotechnology have been selected by the consortium and will be studied under two coordinated approaches:

taxonomy and standardization of culture collection aspects;
molecular biology (including validation and sampling) and computerization of results.

The potential probes and PCR markers are validated by the different participants with the aim that they are useful for molecular identification of fungi, patent certification and environmental studies.

The collection of strains, their DNA, the sequence data and clones, the probes of PCR primers and the standardized protocols are the practical output to the Community.

Coordinator

Institut d'Higiene et d'Epidemiologie
Address
Rue Juliette Wytsman 14
1050 Brussels
Belgium