Article

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Kingsdown, Bristol, UK.
Medical Mycology (Impact Factor: 1.98). 09/2006; 44(5):389-98. DOI: 10.1080/13693780600564613
Source: PubMed

ABSTRACT Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

0 Bookmarks
 · 
89 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycoparasitism - when one fungus parasitizes another - has been reported to affect Beauveria bassiana and mycorrhizal fungi in the field. However, mycoparasitism of any fungi in the Order Entomophthorales has never been reported before now. The majority of entomophthoralean species persist as resting spores (either zygospores or azygospores) in the environment and dormant entomophthoralean resting spores (whether formed as zygospores or azygospores) are thought to be especially well adapted for survival over long periods due to their thick double walls. Entomophthoralean resting spores can accumulate in the soil as large reservoirs of inoculum which can facilitate the onset and development of epizootics. We report parasitism of azygospores of the gypsy moth pathogen Entomophaga maimaiga caged in soil from southern Ohio by the chytrid fungus Gaertneriomyces semiglobifer. G. semiglobifer had previously been isolated from soil samples from North America, Europe and Australia or horse manure from Virginia. After isolation and identification of G. semiglobifer, azygospores of E. maimaiga exposed to zoospores of G. semiglobifer exhibited high levels of mycoparasitism and G. semiglobifer was subsequently reisolated from mycoparasitized resting spores. We discuss the importance of this finding to the epizootiology of insect diseases caused by entomophthoralean fungi.
    Journal of Invertebrate Pathology 10/2013; · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In 1981, four fungal isolates from hair of the European badger (Meles meles) were examined by Dr Phyllis Stockdale at the Commonwealth Mycological Institute, Kew, and deposited in the UK National Collection of Pathogenic Fungi as an undescribed member of the Trichophyton terrestre complex. The present paper formalizes the complete description of a new ascomycete taxon, Arthroderma olidum following successful recent attempts to re-isolate the same fungus from the soil of Badger holes in South West England. Furthermore, using ribosomal RNA gene sequencing, we show that the asexual form of A. olidum is conspecific with the recently described Trichophyton eboreum1 isolated from a human skin specimen in Germany.
    Medical Mycology 09/2006; 44(5):451-9. · 1.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.
    Journal of Industrial Microbiology 03/2014; · 1.80 Impact Factor

Full-text

View
4 Downloads
Available from
Jul 31, 2014