This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okeke, C. N.
Right arrow Articles by Ogawa, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okeke, C. N.
Right arrow Articles by Ogawa, H.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 2001, p. 101-106, Vol. 39, No. 1
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.1.101-106.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Isolation of an Intron-Containing Partial Sequence of the Gene Encoding Dermatophyte Actin (ACT) and Detection of a Fragment of the Transcript by Reverse Transcription-Nested PCR as a Means of Assessing the Viability of Dermatophytes in Skin Scales

Charles N. Okeke, Ryoji Tsuboi,* Masaaki Kawai, Masataro Hiruma, and Hideoki Ogawa

Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

Received 14 April 2000/Returned for modification 31 May 2000/Accepted 17 October 2000

An internal partial sequence of the gene encoding actin (ACT), 725 to 762 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced. An intron that is 56 to 93 bp in length was located along the ACT fragment of all of the dermatophytes at codon position 301 (-3) (a codon number followed by "-3" indicates that the intron directly follows the codon) with reference to the amino acid sequence of human alpha -smooth muscle actin. A primer pair that annealed to exon sequences flanking the ACT-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-PCR) of dermatophyte ACT mRNA. PCR primer pairs with antisense sequence based on the ACT intron sequence were species specific for dermatophytes, suggesting a potential for use in the identification of dermatophytes. The viability of dermatophytes in skin scales was subsequently assessed by the presence of ACT mRNA in total RNA extracted from a 48-h culture of scale samples in 250 µl of yeast carbon base broth. RT-nested PCR of dermatophyte-infected samples amplified an ACT fragment of the predicted size of 171 bp. The results of viability testing based on ACT mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics.

* Corresponding author. Mailing address: Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-5802-1089. Fax: 81-3-3813-9443. E-mail: tsuboi{at}med.juntendo.ac.jp.

Journal of Clinical Microbiology, January 2001, p. 101-106, Vol. 39, No. 1
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.1.101-106.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Gupta, A. K., Zaman, M., Singh, J. (2008). Diagnosis of Trichophyton rubrum from Onychomycotic Nail Samples Using Polymerase Chain Reaction and Calcofluor White Microscopy. J. Am. Podiatr. Med. Assoc. 98: 224-228 [Abstract] [Full Text]  
  • Binstock, J. M. (2007). Molecular Biology Techniques for Identifying Dermatophytes and Their Possible Use in Diagnosing Onychomycosis in Human Toenail: A Review. J. Am. Podiatr. Med. Assoc. 97: 134-144 [Abstract] [Full Text]  
  • Gao, J., Takashima, A. (2004). Cloning and Characterization of Trichophyton rubrum Genes Encoding Actin, Tri r2, and Tri r4. J. Clin. Microbiol. 42: 3298-3299 [Abstract] [Full Text]  
  • Bleve, G., Rizzotti, L., Dellaglio, F., Torriani, S. (2003). Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products. Appl. Environ. Microbiol. 69: 4116-4122 [Abstract] [Full Text]  
  • Brouta, F., Descamps, F., Monod, M., Vermout, S., Losson, B., Mignon, B. (2002). Secreted Metalloprotease Gene Family of Microsporum canis. Infect. Immun. 70: 5676-5683 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»