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Development of mitochondrial SSU rDNA-based oligonucleotide probes for specific detection of common airborne fungi
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
2003 (English)In: Molecular and Cellular Probes, ISSN 0890-8508, Vol. 17, no 6, 281-288 p.Article in journal (Refereed) Published
Abstract [en]

In this study we sequenced partial mitochondrial small subunit rDNA from 32 fungal strains representing 31 species from 16 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence alignment showed several conserved and highly variable regions. The variable regions were deployed to design oligonucleotide probes for each fungal species. The specificity of the designed probes was first examined through homology search against GenBank database then further verified through hybridization experiments to 38 fungal strains. A total of 23 probes were verified as specific to 15 fungal species commonly detected in living and working environments. These new probes will have potential applications in clinical diagnosis and public health-related environmental monitoring.

Place, publisher, year, edition, pages
2003. Vol. 17, no 6, 281-288 p.
Identifiers
URN: urn:nbn:se:umu:diva-4870DOI: 10.1016/S0890-8508(03)00067-7PubMedID: 14602478OAI: oai:DiVA.org:umu-4870DiVA: diva2:144135
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2009-11-25Bibliographically 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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