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Development of molecular techniques for fungal diagnostic research
Umeå University, Faculty of Medicine, Molecular Biology.
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 [en]
Molecular biology, Fungi, DNA markers, Aerosols, Detection and quantification, Environmental monitoring
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-656ISBN: 91-7305-994-3 (print)OAI: oai:DiVA.org:umu-656DiVA: diva2:144140
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
List of papers
1. Development of mitochondrial SSU rDNA-based oligonucleotide probes for specific detection of common airborne fungi
Open this publication in new window or tab >>Development of mitochondrial SSU rDNA-based oligonucleotide probes for specific detection of common airborne fungi
2003 (English)In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, 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.

Identifiers
urn:nbn:se:umu:diva-4870 (URN)10.1016/S0890-8508(03)00067-7 (DOI)14602478 (PubMedID)
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2017-12-14Bibliographically approved
2. Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi.
Open this publication in new window or tab >>Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi.
2004 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 237, no 1, 79-87 p.Article in journal (Refereed) Published
Abstract [en]

Fungi exist in every indoor and outdoor environment. Many fungi are toxigenic or pathogens that may cause various public health concerns. Rapid and accurate detection and identification of fungi require specific markers. In this study, partial mitochondrial large subunit rDNA was amplified and sequenced from 32 fungal strains representing 31 species from 14 genera. Based on the sequence variation pattern, 26 oligonucleotide probes were designed for their discrimination. The specificity of the probes was evaluated through homology search against GenBank database and hybridization examination on 38 fungal strains. The 26 probes were verified as highly specific to 20 fungal species. A two-step detection procedure through PCR followed by probe hybridization gave ten-fold increase in detection sensitivity than single-step PCR assay and would be a practical approach for environmental sample screening. The probes developed in this study can be applied in clinical diagnosis and environmental monitoring of fungal agents.

Keyword
Air Microbiology, Base Sequence, Conserved Sequence, DNA; Fungal/analysis/chemistry, DNA; Mitochondrial/*genetics, DNA; Ribosomal/*analysis, Fungi/*genetics/*isolation & purification, Genes; rRNA, Molecular Sequence Data, Nucleic Acid Hybridization, Oligonucleotide Probes, Polymerase Chain Reaction, RNA/genetics, Sensitivity and Specificity, Sequence Alignment, Sequence Analysis; DNA
Identifiers
urn:nbn:se:umu:diva-17912 (URN)10.1111/j.1574-6968.2004.tb09681.x (DOI)15268941 (PubMedID)
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2017-12-14Bibliographically approved
3. Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR
Open this publication in new window or tab >>Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR
2005 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 5, 65- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. RESULTS: We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. CONCLUSION: The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.

Identifiers
urn:nbn:se:umu:diva-4872 (URN)10.1186/1471-2180-5-65 (DOI)16280082 (PubMedID)
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2017-12-14Bibliographically approved
4. Detection and quantification of Wallemia sebi in aerosols by real-time PCR, conventional PCR, and cultivation.
Open this publication in new window or tab >>Detection and quantification of Wallemia sebi in aerosols by real-time PCR, conventional PCR, and cultivation.
2004 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, 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.

Keyword
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:nbn:se:umu:diva-16826 (URN)10.1128/AEM.70.12.7295-7302.2004 (DOI)15574929 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2017-12-14Bibliographically approved
5. Detection and quantification of Cladosporium in aerosols by real-time PCR.
Open this publication in new window or tab >>Detection and quantification of Cladosporium in aerosols by real-time PCR.
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.

Keyword
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:nbn:se:umu:diva-16825 (URN)10.1039/b509515h (DOI)16395473 (PubMedID)
Available from: 2007-10-12 Created: 2007-10-12 Last updated: 2017-12-14Bibliographically approved

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