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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Methodology for the fast design of fungal DNA probes and PCR primers

Background and objectives

Fungi are of major economic importance as opportunistic pathogens and spoilage organisms but they also have a number of positive uses in food and non-food industries. These useful fungi may be modified genetically to improve their efficiency. To ensure the biosafety of these genetically modified organisms, European regulations require the development of tests for their identification and detection. These tests generally involve the use of unambiguous DNA probes and PCR markers. This aim of this project was to assess methodology for the rapid design of tools for the identification and detection of genetically modified fungi.


Approach and methodology

The groups working on this project combined DNA amplification techniques (RAPD and PCR), sequencing, sequence analysis and taxonomic aspects with a view to identifying regions of the genome that could be used to generate genus and/or species-specific probes and exploring the potential of amplified DNA polymorphisms for taxonomic pre-screening and strain certification. Two types of genomic target were selected: ubiquitous rRNA gene sequences and DNA polymorphisms generated by PCR with arbitrary primers. Co-operation between mycologists and molecular biologists was encouraged and methods were standardised in the participating laboratories.


Main findings and outcome

Several DNA techniques were adapted to fungi and standardised using fungal genera and species used in several areas of biotechnology. A quality assurance structure was set up for strain certification, primers and buffer distribution and a library of DNA from certified strains established. Two quick DNA preparation methods were developed, one involving freeze-thawing, phenol-chloroform extraction and DNA precipitation and the other involving disruption with glass beads in the presence of phenol and SDS followed by DNA precipitation. Both protocols produced DNA of sufficiently high quality for use in PCR, RAPD and direct sequencing. In the standardised PCR protocol, primers of the NS series worked with all the fungi tested whereas only the ITS4 and ITS5 primers were truly universal. The 18S region was found to have little potential for the generation of species-specific DNA probes and PCR primers. In contrast, the ITS adjacent to the 5.8S subunit sequence were found to have great potential for the development of family-, genus- and species-specific probes. Probes were designed using these sequences but were found to have limited potential for differentiating between fungal species. PCR analysis of DNA polymorphism at multiple genetic loci was used to characterise subspecies. The discrimination potential at each locus was found to be independent of the G+C content of the genome. Combination of the multilocus data increased the discrimination potential of the method up to or beyond that of RFLP analysis. A rationale was developed for linking genotypes, genotype population frequencies and loci, making it possible to construct maps of intraspecific diversity.


Conclusions

A standardised methodology based on a combination of classical and high-tech DNA amplification techniques was developed for the assessment of inter- and intra-specific genetic diversity at multiple loci and phylogenic analysis based on DNA sequencing of the ITS regions of fungal ribosomal RNA genes. These methods have potential applications in agrofood industry and research, medical and veterinary research, clinical diagnostics, biosafety and intellectual property protection. Although they were specifically adapted to fungi, similar techniques could also be used for other types of organism.

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Contract number
BIOT-CT91-0301

Period
December 1991 – November 1993

Coordinator
W. Moens
Institut d’Hygiène et d’Epidémiologie (IHEM)
Bruxelles (BE)

 
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Partners


N. Nolard-Tintigner
Institut d’Hygiène et d’Epidémiologie (IHEM)
Bruxelles (BE)

V. Rubio
Universidad Autónoma de Madrid (ES)

J. Frisvad
Technical University of Denmark
Lyngby (DK)

F. Gannon
European Molecular Biology Organization
Heidelberg (DE)

O.F. Rasmussen
Biotechnological Institute
Lyngby (DK)

 
 
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