Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections

Ferrer, Consuelo; Colom, Francisca; Frasés, Susana; Mulet, Emilia; Abad, José Luís; Alió, Jorge L.

doi:10.1128/jcm.39.8.2873-2879.2001

Cited by 293 publications

(195 citation statements)

References 46 publications

(34 reference statements)

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“…This indicates that the ability of the primer mix to amplify a specific DNA fragment from the template DNA is the same as the species-specific primer pair. In contrast, the sensitivity of nested PCR-targeting rDNA of A. fumigatus was found to be 1 to 10 fg (7,26). These results indicate that the sensitivity of our nested PCR system is lower than that of PCR-targeting rDNA.…”

Section: Resultsmentioning

confidence: 99%

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Kanbe

Yamaki

Kikuchi

2002

Microbiology and Immunology

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For PCR‐based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species‐specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species‐specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species‐specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.

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Section: Resultsmentioning

confidence: 99%

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Kanbe

Yamaki

Kikuchi

2002

Microbiology and Immunology

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“…The primers used for the first PCR were ITS1 (5 0 -TCCGTAGGTGAACCTGCGG-3 0 ), ITS4 (5 0 -TCCTCCGCTTATTGATATGC-3 0 ), 14 and for the second semi-nested PCR were ITS3 (5 0 -GCATCGATGA AGAACGCAGC-3 0 ) 15 or ITS86 (5 0 -GTGAATCATCGA ATCTTTGAAC-3 0 ) 14 and ITS4. The primer-binding sites are illustrated in Figure 1.…”

Section: Dna Extraction Pcr and Sequencingmentioning

confidence: 99%

Prospective comparison between conventional microbial work-up vs PCR in the diagnosis of fungal keratitis

Tananuvat

Salakthuantee

Vanittanakom

et al. 2012

Eye

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Purpose The PCR was compared with routine microbial studies for the detection of fungal pathogens in clinically suspected fungal keratitis. Methods A prospective nonrandomized study was undertaken at a tertiary eye care centre to evaluate 30 eyes of 30 patients with presumed fungal keratitis, both fresh and treated. Corneal scrapings were performed on each patient. The specimens were analysed by a semi-nested PCR assay using fungalspecific primers. PCR products were cloned and sequenced for identification, and compared with a conventional microbial work-up (smear and culture). Results Of the 30 samples, the PCR showed positivity in 93.3%, culture in 40%, and potassium hydroxide in 20%. Of the 28 PCR-positive cases, 12 were culture-positive and 16 were culture-negative. Two samples were both PCR and culture test negative. Culture-negative samples were PCR-positive in 16 of 18 (88.9%) cases. The PCR did not yield any false-negative findings in a culturepositive specimen. Both common and uncommon aetiologic fungi have been identified by DNA sequencing analysis. Conclusion The PCR was able to detect fungal DNA in a high proportion of culturenegative cases. Technical considerations of the PCR process include extraction of artifacts and amplification of non-pathogenic DNA. Nonetheless, our findings suggest that the PCR can be a useful adjunct to smear and culture in the rapid diagnosis of fungal keratitis, particularly in cases of failed detection from routine procedures.

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“…High molecular weight DNA was extracted, according to Ferrer et al 5 , and the molecular type was identified by URA5-RFLP, according to Meyer et al 2 . The RFLP patterns were assigned visually by comparison with the patterns obtained from the reference strains.…”

Section: Introductionmentioning

confidence: 99%

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case

Pinto

Pone

et al. 2010

Rev. Soc. Bras. Med. Trop.

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Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas.

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Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections

Cited by 293 publications

References 46 publications

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Identification of the Pathogenic Aspergillus Species by Nested PCR Using a Mixture of Specific Primers to DNA Topoisomerase II Gene

Prospective comparison between conventional microbial work-up vs PCR in the diagnosis of fungal keratitis

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case

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