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Detection and quantification of Wallemia sebi in aerosols by real-time PCR, conventional PCR, and cultivation.
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Rasmuson)
Umeå University, Faculty of Medicine, Molecular Biology. (Rasmuson)
2004 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, Vol. 70, no 12, 7295-7302 p.Article in journal (Refereed) Published
Abstract [en]

Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 10(7) m(-3) by real-time PCR and 10(6) m(-3) by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.

Place, publisher, year, edition, pages
2004. Vol. 70, no 12, 7295-7302 p.
Keyword [en]
Agriculture, Air Microbiology, Ascomycota/classification/genetics/growth & development/*isolation & purification, Colony Count; Microbial, Culture Media, DNA Primers, Molecular Sequence Data, Polymerase Chain Reaction/*methods, Sensitivity and Specificity, Sequence Analysis; DNA, Species Specificity, Spores; Fungal/isolation & purification, Time Factors
Identifiers
URN: urn:nbn:se:umu:diva-16826DOI: 10.1128/AEM.70.12.7295-7302.2004PubMedID: 15574929OAI: oai:DiVA.org:umu-16826DiVA: diva2:156499
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2009-11-24Bibliographically approved
In thesis
1. Development of molecular techniques for fungal diagnostic research
Open this publication in new window or tab >>Development of molecular techniques for fungal diagnostic research
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fungi are present everywhere in indoor and outdoor environments. Many fungi are toxigenic or pathogenic that may cause various public health concerns. Rapid detection, quantification and characterization of fungi in living and working environments are essential for exposure risk assessment to safe guard public health.

Rapid and accurate detection and identification of fungi using molecular method require specific markers. In this thesis, partial mt SSU and LSU rDNA were amplified and sequenced from 31 fungal species of 16 genera. Sequence alignments showed that fungal mt SSU and LSU rDNA contained sufficient amount of variation for the development of markers that can discriminate even among closely related species. Forty-eight probes were designed and were verified as highly specific to 25 fungal species commonly detected in living and working environments. These specific probes would have potential applications in clinical diagnosis and public health-related environmental monitoring.

Nested PCR is a highly sensitive and specific method. Based on the nuclear 18S rDNA sequence variation pattern, three nested PCR systems were developed to detect the conifer tree pathogen Gremmeniella abietina, an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The three nested PCR systems showed high specificity and sensitivity. These methods could have broad applications in forest protection and disease management programs.

Quantitative real-time PCR offers the ability of simultaneous detection and quantification of DNA of a specific microbe in one reaction. Based on the 18S rDNA sequence, two real-time PCR assays were developed to detect and quantify Wallemia sebi, a deuteromycete fungus commonly found in agricultural environments and is suspected to be a causative agent of farmer’s lung disease. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. Application of the real-time PCR methods in the quantification of W. sebi in the aerosols of a farm revealed a high concentration of W. sebi spores (107/m3). The study indicates that W. sebi is a dominant fungus in agriculture environments.

Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. A TaqMan probe and a SYBR Green I based real-time PCR assay were developed to detect and quantify Cladosporium in aerosols. The two real-time PCR systems proved to be highly specific and sensitive for Cladosporium. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. High spore concentration of Cladosporium (107/m3) was observed in a cow barn. Cladosporium spore concentration in paper and pulp factory and countryside house also exceeded threshold value for clinical significance. Prolonged exposure in these environments could impose certain health risk. Thus, monitoring Cladosporium spore concentration in indoor environments is important for indoor air quality control.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2005. 75 p.
Keyword
Molecular biology, Fungi, DNA markers, Aerosols, Detection and quantification, Environmental monitoring, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-656 (URN)91-7305-994-3 (ISBN)
Public defence
2006-01-18, Stora föreläsningssalen, Arbetslivsinstitutet, Petrus Laestadius väg, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2009-11-24Bibliographically approved

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Publisher's full textPubMedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=15574929&dopt=Citation

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Rasmuson-Lestander, Åsa

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