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Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

ABSTRACT: BACKGROUND: Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. METHODS: The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. RESULTS: The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum) and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. CONCLUSION: It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.

SUBMITTER: Rezaei-Matehkolaei A

PROVIDER: S-EPMC3481703 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

Rezaei-Matehkolaei A A Makimura K K Shidfar Mr M Zaini F F Eshraghian Mr M Jalalizand N N Nouripour-Sisakht S S Hosseinpour L L Mirhendi H H

Iranian journal of public health 20120331 3

<h4>Background</h4>Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utili ...[more]

PMID: 23113152

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Project description:BackgroundDNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.Methodology/principal findingsThe purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.Conclusions/significanceThe ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.

Dataset Information

Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

Publications

Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

Similar Datasets

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