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  • IV — Inventaire, Caractérisation et Suivi de la Diversité Microbienne / Inventory, Characterization and Monitoring of Microbial Diversity
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Use of specific DNA probes for the rapid characterization of yeasts isolated from complex biotopes

Utilisation de sondes ADN spécifiques pour la caractérisation rapide de levures isolées d’écosystèmes complexes

Genetics Selection Evolution volume 33, Article number: S353 (2001) Cite this article

Abstract

We describe a rapid method developed to identify yeast species commonly found during cheese ripening. It is based on the isolation of species-specific sequences and their hybridization with coarse preparations of genomic DNA. Several strategies were followed for the construction of probes: PCR amplification of sequences available in databases, random cloning of genomic DNA fragments, specific RAPD fragments and PCR-amplified ribosomal DNA fragments. After validation, the probes were applied to the characterization of 400 yeast strains isolated from various French cheeses. Since the strains had been previously identified with classical diagnostic tests, we were able to compare molecular and conventional identification. In addition, the specific probes for one of the species, Debaryomyces hansenii, were used successfully in colony hybridization experiments. The probes developed here proved to be very useful for the screening of large yeast collections and for the assessment of biodiversity within the yeast flora in cheese.

Résumé

Au cours de ce travail, nous avons développé une méthode à la fois rapide et sensible pour l’identification des levures couramment isolées pendant l’affinage du fromage. Son principe repose sur l’hybridation de séquences spécifiques sur les préparations brutes d’ADN génomique des souches à identifier. Pour la construction des sondes, nous avons dû suivre plusieurs stratégies différentes: amplification de séquences disponibles dans les bases de données, clonage au hasard de séquences d’ADN génomique, isolement de séquences spécifiques obtenues par amplification RAPD d’ADN génomique ou par amplification d’ADNr. Après leur validation, les sondes ont pu être mises en œuvre dans différentes applications. Elles ont été utilisées pour cribler 400 souches de levures isolées de différents fromages français. Les souches ayant été préalablement identifiées par des tests classiques, nous avons pu comparer les deux types de méthodes d’identification, moléculaire et conventionnelle. La sonde spécifique de D. hansenii a été testée avec succès dans des essais d’hybridation sur colonie. Au total, ces sondes se sont révélées être de bons outils pour le suivi et l’inventaire de la biodiversité des levures fromagères.

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Authors and Affiliations

  1. Collection de levures d’intérêt biotechnologique, Génétique moléculaire et cellulaire, Cnrs URA1925, Inra UMR216, Institut national agronomique Paris-Grignon, 78850, Thiverval-Grignon, France

    Anne-Marie Davila, Monique Diez, Mauricio Corredor, Yves Pagot, Michèle Winkler, Claude Gaillardin & Serge Casaregola

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  1. Anne-Marie Davila
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  2. Monique Diez
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  3. Mauricio Corredor
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  4. Yves Pagot
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  5. Michèle Winkler
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  6. Claude Gaillardin
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  7. Serge Casaregola
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Correspondence to Serge Casaregola.

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Davila, AM., Diez, M., Corredor, M. et al. Use of specific DNA probes for the rapid characterization of yeasts isolated from complex biotopes. Genet Sel Evol 33 (Suppl 1), S353 (2001). https://doi.org/10.1186/BF03500889

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Genetics Selection Evolution

ISSN: 1297-9686