Korean J Med Mycol.
2011 Sep;16(3):86-89.
PCR-reverse Blot Hybridization Assay for Species Identification of Dermatophytes
- Affiliations
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- 1Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju , 220-710, Korea. hyelee@yonsei.ac.kr
- 2M&D, Inc., Wonju Eco Environmental Technology Center, Wonju, 220-710, Korea.
- 3Korean Culture Collection of Medical Fungi (KCMF), College of Medical Science, Konyang University, Daejeon, 302-718, Korea.
- 4Department of Laboratory Medicine, Samsung Medical Center, Seoul, 135-710, Korea.
- 5Department of Dermatology, Medical School of Chonnam National University, Gwangju, 140-755, Korea.
- 6Department of Dermatology, Konkuk University School of Medicine, Seoul, 143-914, Korea.
- 7Department of Biomedical Laboratory Science, College of Medical Science, Konyang University, Daejeon, 302-718, Korea. ykkim3245@konyang.ac.kr
Abstract
- BACKGROUND
Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) cause cutaneous mycoses called dermatophytosis. Forproper anti-dermatophytosis therapy, accurate and early diagnosis of dermatophytes is important. Laboratory diagnosis of dermatophytosis for dermatophytes still relies on microscopic and macroscopic examination of in vitro cultures and some physiological tests. These methods (conventional methods) are time-consuming (2~4 weeks) and yet, still have low sensitivity and specificity. Recently, in order to overcome such limitations of conventional methods, molecular-based methods have been developed to identify dermatophytes. The polymerase chain reaction-reverse blot hybridization assay (PCR-REBA) allows sensitive and specific identification of dermatophytes species.
OBJECTIVE
This study was aimed to develop a new PCR-REBA with higher sensitivity using less amount of probe concentration, so the assay can be more practical in clinical settings.
METHODS
For this, PCR primers and species-specific oligonucleotide probes were designed within the internal transcribed sequences 1 region between 5.8S and 18S rRNA. The species-specific probes designed in this study was to identify 6 species (T. rubrum, T. mentagrophytes, T. tonsurans, M. canis, M. gypseum, and E. floccosum) comprised 99% of dermatophytes isolatedin Korea.
RESULTS
The detection efficiency of the PCR-REBA was compared with the microscopic method, and the results showed that the sensitivity of the PCR-REBA developed in this study is 100 times higher than previously developed one. Subsequently, the results of PCR-REBA were evaluated using clinical isolates. DNAs from a total of 68 clinical isolates were analyzed by PCR-REBA, and the inconsistent results between PCR-REBA and conventional microscopic identification results were confirmed by sequence analysis.
CONCLUSION
In brief, the results showed that results of sequence analysis were identical with PCR-REBA implying newly developed PCR-REBA is very useful method for accurate and rapid identification of dermatophytes and would provide higher simplicity, specificity, sensitivity than conventional method.
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