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Detection and quantification of Cladosporium in aerosols by real-time PCR.
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Rasmuson)
Umeå University, Faculty of Medicine, Molecular Biology. (Lestander)
2006 (English)In: Journal of Environmental Monitoring, ISSN 1464-0325, E-ISSN 1464-0333, Vol. 8, no 1, 153-160 p.Article in journal (Refereed) Published
Abstract [en]

Cladosporium is one of the most common airborne molds found in indoor and outdoor environments. Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. To accurately quantify the levels of Cladosporium in indoor and outdoor environments, two real-time PCR systems were developed in this study. The two real-time PCR systems are highly specific and sensitive for Cladosporium detection even in a high background of other fungal DNAs. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. The investigation revealed a high spore concentration of Cladosporium (10(7) m(-3)) in a cow barn that accounted for 28-44% of the airborne fungal propagules. In a countryside house that uses firewood for heating and in a paper and pulp factory, Cladosporium was detected at 10(4) spores m(-3), which accounted for 2-6% of the fungal propagules in the aerosols. The concentrations of Cladosporium in these three indoor environments far exceeded the medical borderline level (3000 spores m(-3)). In a power station and a fruit and vegetable storage, Cladosporium was found to be a minor component in the aerosols, accounted for 0.01-0.1% of the total fungal propagules. These results showed that monitoring Cladosporium in indoor environments is more important than in outdoor environments from the public health point of view. Cladosporium may not be the dominant fungi in some indoor environments, but its concentration could still be exceeding the threshold value for clinical significance. The methods developed in this study could facilitate accurate detection and quantification of Cladosporium for public health related risk assessment.

Place, publisher, year, edition, pages
2006. Vol. 8, no 1, 153-160 p.
Keyword [en]
Aerosols/analysis, Air Microbiology, Air Pollution; Indoor/*analysis, Cladosporium/genetics/*isolation & purification, Colony Count; Microbial, DNA; Fungal/analysis, DNA; Mitochondrial/analysis, DNA; Ribosomal/analysis, Environmental Monitoring/methods, Polymerase Chain Reaction, Sequence Alignment, Sequence Analysis; DNA
Identifiers
URN: urn:nbn:se:umu:diva-16825DOI: 10.1039/b509515hPubMedID: 16395473OAI: oai:DiVA.org:umu-16825DiVA: diva2:156498
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2017-12-14Bibliographically 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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