Detection and identification of fungi in blood using broad‐range 28S rDNA PCR amplification and species‐specific hybridisation

Evertsson, U; Monstein, Hans‐Jürg; Johansson, Amanda

doi:10.1034/j.1600-0463.2000.d01-73.x

Cited by 32 publications

(24 citation statements)

References 29 publications

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“…Detection of oligonucleotide probe hybridization has been reported using microtitration plate-based enzyme immunoassay (5,17,30), Southern or slot blotting (4,6,23,28), and fluorogenic probes (8,19,24). We chose the RLB format, given the advantages of relative simplicity, low cost, ready availability of the materials and the methods, and ability to simultaneously analyze multiple specimens against multiple probes (7).…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

et al. 2006

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We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit crosshybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10 0.5 cells/ml and 10 2 conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.

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Section: Discussionmentioning

confidence: 99%

Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

et al. 2006

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“…Given that more than 200 fungal species have been reported to cause disease in humans and companion animals (11), the clinical utility of a species-specific or even a genus-specific assay is limited. Panfungal PCR assays, on the other hand, have the potential to detect all fungal species, but many rely on additional, time-consuming techniques, such as species-specific probes and hybridization, to identify the pathogen (10,12,15,25,29,34,37). Furthermore, probe design is restricted to known pathogens and does not allow the identification of new and emerging agents.…”

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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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“…A number of studies have described probes, restriction fragment length polymorphism, or other methods to identify unique ribosomal DNA (rDNA) sequences (10, 16-18, 22, 23, 32, 40, 41, 43). The most common approaches have targeted portions of the rDNA of species of Candida (3,8,10,31,38,44). Although these published PCR methods have been useful for the identification of fungal species, they either identify only one species at a time or require a probe hybridization procedure that incurs time and expense.…”

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confidence: 99%

Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

2002

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A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.

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Detection and identification of fungi in blood using broad‐range 28S rDNA PCR amplification and species‐specific hybridisation

Cited by 32 publications

References 29 publications

Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

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

Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

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