Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis

Velegraki, Aristea

doi:10.1016/s0928-8244(98)00150-3

Cited by 29 publications

(15 citation statements)

References 21 publications

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“…It could be increased if more skin scales were used from parasitized specimens, but this was not always feasible, especially when sampling small and scanty lesions. However, the relative sensitivity of 10 AE 5 yeast cells, as estimated using pure cultures, matches that previously reported for Candida species in spiked biological fluids [11,12].…”

Section: Discussionsupporting

confidence: 86%

“…In these instances, nested PCR using the ITS 1/4 and ITS 3/4 primers can be useful in the direct detection and identification of Malassezia species. The benefit of using general fungal primers was the co-detection of non-Malassezia species in patient skin scales, for which ITS PCR-RFLP patterns are already known for many emerging opportunistic fungi [11,12,[27][28][29][30].…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, to achieve adequate yeast cell lysis and sufficient DNA purity for PCR amplification, a modification of the method proposed by the manufacturers of the Silica extraction kit (MBI, Fermentas, Lithuania) was incorporated into the extraction protocol as follows. A standard loopful of volume capacity 10 À3 (Greiner GmbH, Germany) of yeast pure culture was suspended in 100 ml lysis buffer [(200 mM Tris-HCl, pH 8, 250 mM NaCl, 25 mM ethylenediamine-tetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) (all from Sigma)] as previously described [11]. Cells were disrupted mechanically with an orbital homogenizer, following addition of a further 350 ml lysis buffer.…”

Section: Dna Extraction From Pure Culturesmentioning

confidence: 99%

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Identification of Malassezia species from patient skin scales by PCR-RFLP

Gaitanis

Velegraki

Frangoulis³

et al. 2002

Clinical Microbiology and Infection

100

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Dna Extraction From Pure Culturesmentioning

confidence: 99%

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Identification of Malassezia species from patient skin scales by PCR-RFLP

Gaitanis

Velegraki

Frangoulis³

et al. 2002

Clinical Microbiology and Infection

100

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“…

Lass-Flörl 2009) with a mortality rate ranging from 50 to 80%, (Fraser et al 1992). In the absence of pathognomonic signs or symptoms, diagnosis of invasive candidiasis has to be made after isolating and identifying yeast species by morphology and assimilation tests that usually take several days (Velegraki et al 1999). Furthermore,

AbstractAims: This work focuses on the development of a method for the identification of pathogenic yeast.

…”

mentioning

confidence: 99%

Identification of pathogenic yeast species by polymerase chain reaction amplification of theRPS0gene intron fragment

Martínez

Gómez

Pemán

et al. 2009

Journal of Applied Microbiology

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Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (107–105 cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.

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“…The rRNA genes have been extensively used as the targets for molecular identification [12,13]. These methods include DNA probes [14], PCR-restriction enzyme analysis [15], real-time PCR [16], and DNA sequencing [17,18]. PCR techniques are particularly promising because of their simplicity, sensitivity, and specificity.…”

Section: Introductionmentioning

confidence: 99%

Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

Hung

Shiu

et al. 2011

BMC Infect Dis

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BackgroundAirborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate.MethodsWe developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies.ResultsA collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26) and 97.7% (43/44), respectively.ConclusionsIdentification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

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Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis

Cited by 29 publications

References 21 publications

Identification of Malassezia species from patient skin scales by PCR-RFLP

Identification of Malassezia species from patient skin scales by PCR-RFLP

Identification of pathogenic yeast species by polymerase chain reaction amplification of theRPS0gene intron fragment

Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

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