Molecular probes for diagnosis of fungal infections

Sandhu, Gurpreet S.; Kline, Bruce C.; Stockman, Leslie; Roberts, Glenn D.

doi:10.1128/jcm.33.11.2913-2919.1995

Cited by 353 publications

(137 citation statements)

References 18 publications

(2 reference statements)

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“…Although phylogenetic analysis is impractical for routine verification of molecular taxonomy results in the clinical laboratory, it was used here to corroborate homology (BLAST and FASTA)-based identification, and to assess the phylogenetic inference of the D2 region of the 25-28S rRNA gene. Our system exploits a D2 region shorter than the fragment used in the commercial MicroSeq kit, which is defined by the P3-U2 primer pair 29 and extends 70 bp upstream of the U1 forward primer used by us ( Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

“…Polymerase chain reaction was performed with either yeast thermolysates or purified genomic DNA. Universal primers designed on the highly conserved regions of the Saccharomyces cerevisiae 25S rDNA LSU, 29 flanking the D2 variable region at the 5¢ end of LSU 30,31 (Fig. 1), were used to amplify DNA fragments corresponding to the 260 bp D2 fragment of the S. cerevisiae…”

Section: Pcr Amplificationmentioning

confidence: 99%

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Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Putignani

Paglia

Bordi

et al. 2008

Mycoses

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Clinically relevant yeasts are conventionally identified by a combination of phenotypic tests, which occasionally provide ambiguous results for atypical isolates or uncommon species. In this study, we evaluate a direct polymerase chain reaction-sequencing method, which exploits sequence divergence in the hypervariable D2 region of the large subunit of the 25-28S ribosomal RNA (rRNA) gene for identification of facultative pathogenic asco- and basidiomycota. A panel of 53 yeasts, including 40 clinical isolates and 13 reference strains representative of some clinically relevant taxa, was investigated by combining standard phenotypic tests with commercial identification systems (RapID, API 20C AUX), and results were compared with the taxonomic allocations inferred by D2 sequence analysis. Species-level resolution was achieved for almost all (52/53) strains by combining internet-based D2 sequence homology (BLAST and FASTA) searches in free-access synchronised databases with phylogenetic analysis. The phylogenetic information carried by the short D2 sequence substantiates a pattern of molecular evolution, which is similar to that inferred from analysis of the larger D1/D2 region, and consistent with previously published 25-28S rRNA phylogenetic architectures of facultative pathogenic yeast, including recently identified species. Inconsistency between conventional and molecular identification results was observed for 11/53 strains, likely on account of the ambiguous interpretation of phenotypic tests.

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Section: Discussionmentioning

confidence: 99%

Section: Pcr Amplificationmentioning

confidence: 99%

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Putignani

Paglia

Bordi

et al. 2008

Mycoses

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“…1 A portion of the 28S rDNA from the brain isolate was also amplified by PCR using universal fungal primers U1 and U2 as previously described. 2 Nucleotide sequencing of amplicons was carried out by a commercial company (GENEWIZ, Inc., South Plainfield, NJ, USA). Sequences were confirmed by sequencing both strands.…”

Section: Case Presentationmentioning

confidence: 99%

Disseminated protothecosis diagnosed by evaluation of CSF in a dog

Lane¹,

Meinkoth²,

Brunker

et al. 2012

Veterinary Clinical Pathol

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A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.

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“…Nevertheless, according to Dorn-In et al (2013), there are some universal primer pairs such as ITS1F/ITS4 (Gardes & Bruns, 1993), U1/U2 (Sandhu et al, 1995) and ITS1/ITS5.8R that amplify only DNA from fungi but not from plants. The latter primer pair was used successfully to reconstruct and to identify fungal flora in heatprocessed meat products, which were already spiced (Dorn-In et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR

Dorn‐In¹,

Fahn²,

Hölzel³

et al. 2014

FEMS Microbiol Lett

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Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10 CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10 CFU g(-1) culturable fungi, while there was < 2.83 log10 CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.

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Molecular probes for diagnosis of fungal infections

Abstract: We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus,

Cited by 353 publications

References 18 publications

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Identification of clinically relevant yeast species by DNA sequence analysis of the D2 variable region of the 25–28S rRNA gene

Disseminated protothecosis diagnosed by evaluation of CSF in a dog

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR

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