Identification of Dermatophytes based on polymorphysms of nuclear and mitochondrial ribosomal DNA regions

• Project/Area Number: 10670811
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Dermatology
• Research Institution: Kanazawa Medical University
• Principal Investigator: MOCHIZUKI Takashi Kanazawa Medical University, Department of Dermatology, Associate, 医学部, 助教授 (80190958)
• Project Period (FY): 1998 – 2000
• Project Status: Completed (Fiscal Year 2001)
• Budget Amount: ¥2,800,000 (Direct Cost: ¥2,800,000); Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: Dermatophytes / Identification / Molecular technique / ribosomal DNA / ITS / リボゾームDNA

Research Abstract

The present study was conducted to evaluate the efficacy of molecular techniques for the identification of dermatophytes isolated from human lesions. The techniques used in the study were, restriction fragment length polymorphisms (RFLP) of ribosomal (r) DNA regions in mitochondrial (mt) DNA, RFLP analysis of spacer regions of rDNA in genomic DNA, and sequence analysis of internal transcribed spacer (ITS) of rDNA in genomic DNA.
Using RFLP of PCR amplicons of rDNA region of mtDNA, the profiles generated by digestion with restriction enzyme HinA III showed differences between T. rubrum and T. mentagrophytes. But there was no difference between the RFLP profiles between these two species when the amplicons were digested with Hae III and Msp I. The technique could be used for the differenciation between T. rubrum and T. mentagrophytes, but the information obtained by the technique was rather minimum.
There were many advantages of sequence analysis of ITS1 of rDNA in genomic DNA. The method was highly reproducible and highly sensitive. Moreover, once the sequence is determined, the data could be used elsewhere and referred to data deposited in gene banks. The expense of the method was, however, the drawback in the application in practice.
PCR-RFLP analysis of ITS regions were performed on several species in the T. mentagrophytes complex, T. rubrum and E. floccosum. Digestion of ITS 1+2 regions (ca. 700 bp) by Mva I or Hint. I indicated these profiles to be species specific. The sensitivity and reproducibility was enough for the species level identification. Also the technique was inexpensive and time saving, therefore, the method is also suitable for treatment of a number of isolate at the same time.
In conclusion, I propose to use PCR-RFLP analysis of ITS 1+2 regions by Mva I or Hinf I for the identification of dermatophytes in practice. If the result is unclear or unacceptable, then determine the nucleotide sequence of ITS 1, and refer the data to those deposited in a genebanks.

Research Products

• [Publications] Takashi Mochizuki, et al.: "Identification of several clinical isulates of devoatophytes based on the nucleotidk seguence of Internal transcribed space_r1(ITS 1)in nuclear ribosomal DNA."Journal of Dermatology. 26. 276-281 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Mochizuki,T., Kawasaki,M., Ishizaki,H., Makimura,K.: "The Identification of several clinical isolates of dermatophytes based on the nucleotide sequence if internal transcribed spacer 1)ITS1) in nuclear ribosomal DNA"Journal of Dermatology. Vol. 26. 276-281 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Takashi Mochizuki, et al.: "Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of Internal transcribed spacer 1(ITS 1) in nuclear ribosomal DNA."Journal of Dermatology. 26. 276-281 (1999)
• [Publications] Takashi Mochizuki et al.: "Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of internal transcribed spacer 1 in nuclear ribosomal DNA"The Journal of Dermatology. 26. 276-281 (1999)