Analysis of the internal transcribed spacer regions of ribosomal DNA in common airborne allergenic fungi

Gaskell, Giles J.; Carter, Dee; Britton, Warwick J.; Tovey, Euan R.; Benyon, F. H. L.; Lovborg, U.

doi:10.1002/elps.1150180914

Cited by 27 publications

(12 citation statements)

References 10 publications

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“…and A. lentulus, and Fusarium spp.). These results were not unexpected, since it has been reported that ITS sequence variations among some species of fungi, including Aspergillus spp., are minimal (13,17,19,36), and the differentiation of Fusarium species is complicated by the presence of Ͼ1 ITS sequence variant in a single strain (27). For those species that cannot be discriminated using the ITS1 region alone, future work may involve sequence analysis of additional genes, such as ITS2, the D1-D2 region of 28S rDNA, or intergenic spacer regions (19).…”

Section: Discussionmentioning

confidence: 99%

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

et al. 2007

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Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n ‫؍‬ 38) or solely histologically proven (n ‫؍‬ 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n ‫؍‬ 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.

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Section: Discussionmentioning

confidence: 99%

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

et al. 2007

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“…Biochemical characterizations which have been studied include the detection and identification of secondary metabolites (200), the identification the ubiquinone system (400), and the examination of isoenzyme patterns (369,400,543,554). Molecular data have been obtained on total DNA (105,218,501), mitochondrial DNA (mtDNA) (127,543) or ribosomal DNA (rDNA) (105,127,210,543,618) by using various methodological approaches, mainly restriction fragment length polymorphisms (RFLP) visualized with or without hybridization to specific probes and sequencing of characteristic DNA regions. Criteria which have been suggested as useful in the identification of A. fumigatus are summarized in Table 1.…”

Section: Introductionmentioning

confidence: 99%

“…Amplification by PCR of specific sequences, originally identified by random amplification, seems promising (71). While sequencing of the internally transcribed spacers ITS1 and ITS2 of rDNA has not been completed, there appear to be sufficient differences to distinguish Neosartorya species and A. fumigatus (105,210,502).…”

Section: Introductionmentioning

confidence: 99%

Aspergillus fumigatusand Aspergillosis

1999

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SUMMARY Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.

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“…Hence, such characters are useful for the classification of closely related organisms (9,11,24,25). By using ITS regions (ITS1-5.8S-ITS2), we previously found that single-strand conformation polymorphism (SSCP) analysis assisted in the morphological identification of Section Flavi strains (17).…”

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confidence: 99%

Heteroduplex Panel Analysis, a Novel Method for Genetic Identification of Aspergillus Section Flavi Strains

Kumeda

Asao

2001

Appl Environ Microbiol

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For genetic identification of Aspergillus Section Flavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11 A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. When Penicillium or non-Section Flavi Aspergillus was subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64-

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Analysis of the internal transcribed spacer regions of ribosomal DNA in common airborne allergenic fungi

Cited by 27 publications

References 10 publications

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

Aspergillus fumigatusand Aspergillosis

Heteroduplex Panel Analysis, a Novel Method for Genetic Identification of Aspergillus Section Flavi Strains

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