PCR-reverse Blot Hybridization Assay을 이용한 피부사상균의 동정
PCR-reverse Blot Hybridization Assay for Species Identification of Dermatophytes
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초록
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, molecularbased 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 PCRREBA 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.
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, molecularbased 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 PCRREBA 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.
참고문헌 (22)
- Havlickova B, "Epidemiological trends in skin mycoses worldwide", Mycoses, 51, pp.2~15 (2008)
- 장수정, "국내 표재성 진균증 원인 균종의 변화 추이", 대한의진균학회지, 9(2), pp.91~99 (2004), 대한의진균학회1
- 김기홍, "우리나라에서 시대적 및 사회적 변화에 따른 백선환자의 임상양상의 변화와 원인균의 변천", 대한의진균학회지, 11(1), pp.1~12 (2006), 대한의진균학회1
- Blanco JL, "Immune response to fungal infections", Vet Immunol Immunopathol, 15(125), pp.47~70 (2008)
- Garg J, "Rapid detection of dermatophytes from skin and hair", BMC Res Note, 18, pp.2~6 (2009)
- Jessup CJ, "Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates", J Clin Microbiol, 38, pp.341~344 (2000)
- 신영민, "조갑진균증의 진단에 있어 KOH 검사, 진균 배양 검사, 병리 조직 검사 및중합효소 연쇄반응의 비교", 대한의진균학회지, 12(2), pp.59~69 (2007), 대한의진균학회1
- Gupta AK, "Update in antifungaltherapy of dermatophytosis", Mycopathologia, 166, pp.353~367 (2008)
- Nweze EI, "Agarbased disk diffusion assay for susceptibility testing of dermatophytes", J Clin Microbiol, 48, pp.3750~3752 (2010)
- Robert R, "Conventional methods for the diagnosis of dermatophytosis", Mycopathologia, 166, pp.295~306 (2008)
- Kwon OC, "Significance of polymerase chain reaction for the detection of causative fungi of onychomycosis: comparison with fungus culture and KONCPA (KOH + nail clipping + PAS (periodic acid-schiff) stain)", Korean J Dermatol, 37, pp.1457~1465 (1999)
- Dobrowolska A, "PCR-RFLP analysis of the dermatophytes isolated from patients in central poland", J Dermatol Sci, 42, pp.71~74 (2006)
- Wengenack NL, "Fungal molecular diagnostics", Clin Chest Med, 30, pp.391~408 (2009)
- Hay RJ, "New molecular tools in the diagnosis of superficial fungal infections", Clinics in Dermatology, 28, pp.190~196 (2010)
- Turin L, "Fast, simple and highly sensitive double-rounded polymerase chain reaction assay to detect medically relevant fungi in dermatological specimens", Eur J Clin Invest, 30, pp.511~518 (2000)
- Uchida T, "Comparative study of direct polymerase chain reaction, microscopic examination and culture-based morphological methods for detection and identification of dermatophytes in nail and skin samples", J of Dermatology, 36, pp.202~208 (2009)
- Baek SC, "Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis", Int J Dermatol, 37, pp.682~686 (1998)
- Bergmans AM, "Andriesse GI, Wintermans RG. Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material", Clin Microbiol Infec, 16, pp.704~710 (2010)
- Bergmans AM, "Validation of PCRreverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples", Clin Microbiol Infect, 14, pp.778~788 (2008)
- Martin C, "Development of a PCRbasedlineprobeassay for identification of fungal pathogens", J Clin Microbiol, 38, pp.3735~3742 (2000)
- Bagyalakshmi R, "Application of polymerase chain reaction (PCR) and PCR based restriction fragment length polymorphism for detection and identification of dermatophytes from dermatological specimens", Indian J Dermatol, 53, pp.15~20 (2008)
- De Baere T, "Evaluation of internal transcribed spacer2-RFLP analysis for the identification of dermatophytes", Med Microbiol, 59, pp.48~54 (2010)