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Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions.
J Clin Diagn Res. 2013 Apr; 7(4):646-51.JC

Abstract

PURPOSE

A pan fungal primer targeting the Internal Transcribed Spacer (ITS) region and optimization of PCR-RFLP using a dermatophyte specific primer targeted the 18S ribosomal DNA (rDNA) region were performed for the identification of dermatophyte species and strains directly from clinical specimens.

MATERIALS AND METHODS

One hundred and thirty eight specimens (129 skin scrapings and 9 nail clippings) from clinically suspected cases of dermatophytosis were collected and subjected to direct microscopy and culture. Among them, 66 skin scrapings and 3 nail clippings were processed for genotyping by PCR-RFLP analysis using the Mva I, Hae III and the Dde I restriction enzymes.

RESULTS

Of the 138 specimens, 81 specimens were positive for dermatophytosis, the most common one being Trichophyton rubrum (47), followed by Trichophyton mentagrophytes (25) and Epidermophyton floccosum (9). Of the 47 T. rubrum isolates, 10 were T. rubrum var. raubitschekii which were identified phenotypically as urease positive and by DNA sequencing. Since they exhibited minor morphological and physiological features, they have currently been synonymized with T. rubrum. Of the 25 T. mentagrophytes isolates, three were Trichophyton interdigitale, which were identified by DNA sequencing. Among the 66 skin specimens smear, culture and PCR showed the presence of dermatophytes in 36 (54.54%), 42 (63.63%) and 47 (71.21%) cases respectively. Among the three nail specimens, only one was found to be positive for dermatophytosis by smear, culture and PCR.

CONCLUSION

Amplification of the dermatophyte specific primer is appropriate in the identification of dermatophytes directly from the clinical material. PCR targeting the ITS region by using the Mva I and the Dde I enzymes was equally good for the RFLP analysis. However, by using the above three restriction enzymes, no strain variations were detected among the T. rubrum and the T. mentagrophytes strains.

Authors+Show Affiliations

PhD Research Scholar, Department of Microbiology, Sri Ramachandra University , Chennai, India .No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

23730638

Citation

Elavarashi, Elangovan, et al. "Optimization of PCR-RFLP Directly From the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions." Journal of Clinical and Diagnostic Research : JCDR, vol. 7, no. 4, 2013, pp. 646-51.
Elavarashi E, Kindo AJ, Kalyani J. Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions. J Clin Diagn Res. 2013;7(4):646-51.
Elavarashi, E., Kindo, A. J., & Kalyani, J. (2013). Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions. Journal of Clinical and Diagnostic Research : JCDR, 7(4), 646-51. https://doi.org/10.7860/JCDR/2013/5363.2873
Elavarashi E, Kindo AJ, Kalyani J. Optimization of PCR-RFLP Directly From the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions. J Clin Diagn Res. 2013;7(4):646-51. PubMed PMID: 23730638.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions. AU - Elavarashi,Elangovan, AU - Kindo,Anupma Jyoti, AU - Kalyani,Jagannathan, Y1 - 2013/02/12/ PY - 2012/11/27/received PY - 2013/01/17/accepted PY - 2013/6/5/entrez PY - 2013/6/5/pubmed PY - 2013/6/5/medline KW - DNA sequencing KW - Dermatophytosis KW - Genotyping KW - PCR KW - RFLP SP - 646 EP - 51 JF - Journal of clinical and diagnostic research : JCDR JO - J Clin Diagn Res VL - 7 IS - 4 N2 - PURPOSE: A pan fungal primer targeting the Internal Transcribed Spacer (ITS) region and optimization of PCR-RFLP using a dermatophyte specific primer targeted the 18S ribosomal DNA (rDNA) region were performed for the identification of dermatophyte species and strains directly from clinical specimens. MATERIALS AND METHODS: One hundred and thirty eight specimens (129 skin scrapings and 9 nail clippings) from clinically suspected cases of dermatophytosis were collected and subjected to direct microscopy and culture. Among them, 66 skin scrapings and 3 nail clippings were processed for genotyping by PCR-RFLP analysis using the Mva I, Hae III and the Dde I restriction enzymes. RESULTS: Of the 138 specimens, 81 specimens were positive for dermatophytosis, the most common one being Trichophyton rubrum (47), followed by Trichophyton mentagrophytes (25) and Epidermophyton floccosum (9). Of the 47 T. rubrum isolates, 10 were T. rubrum var. raubitschekii which were identified phenotypically as urease positive and by DNA sequencing. Since they exhibited minor morphological and physiological features, they have currently been synonymized with T. rubrum. Of the 25 T. mentagrophytes isolates, three were Trichophyton interdigitale, which were identified by DNA sequencing. Among the 66 skin specimens smear, culture and PCR showed the presence of dermatophytes in 36 (54.54%), 42 (63.63%) and 47 (71.21%) cases respectively. Among the three nail specimens, only one was found to be positive for dermatophytosis by smear, culture and PCR. CONCLUSION: Amplification of the dermatophyte specific primer is appropriate in the identification of dermatophytes directly from the clinical material. PCR targeting the ITS region by using the Mva I and the Dde I enzymes was equally good for the RFLP analysis. However, by using the above three restriction enzymes, no strain variations were detected among the T. rubrum and the T. mentagrophytes strains. SN - 2249-782X UR - https://www.unboundmedicine.com/medline/citation/23730638/Optimization_of_PCR_RFLP_Directly_from_the_Skin_and_Nails_in_Cases_of_Dermatophytosis_Targeting_the_ITS_and_the_18S_Ribosomal_DNA_Regions_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23730638/ DB - PRIME DP - Unbound Medicine ER -
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