Bagyalakshmi R, Senthilvelan B, Therese K L, Murugusundram S, Madhavan H N
Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, No. 18, College Road, Chennai - 600 006, India.
Indian J Dermatol. 2008 Jan;53(1):15-20. doi: 10.4103/0019-5154.39735.
To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.
Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.
PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.
PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.
开发并优化基于聚合酶链反应的限制性片段长度多态性（PCR-RFLP）方法，该方法靶向真菌的18S核糖体DNA（rDNA）和内转录间隔区（ITS）区域，用于皮肤癣菌的快速检测和鉴定。
使用皮肤癣菌及其他真菌的标准菌株和实验室分离株，对两种靶向真菌18S rDNA和ITS区域的PCR-RFLP方法进行优化。68份皮肤科临床标本（指甲剪屑（56份）、水疱取材（8份）、发根（2份）、足部鳞屑斑块刮屑（1份）以及皮肤科医生采集的皮肤刮屑（1份））同时采用优化后的PCR-RFLP方法和传统真菌学方法（涂片和培养）进行检测。
靶向18S rDNA和ITS区域的PCR分别对10皮克红色毛癣菌DNA和1飞克红色毛癣菌DNA敏感。靶向18S rDNA的PCR对皮肤癣菌具有特异性，随后的RFLP可将其鉴定到种水平。靶向ITS区域的PCR-RFLP可将皮肤癣菌与其他真菌区分开，并鉴定到种水平。在68份检测的临床标本中，两种PCR-RFLP方法均显示27例（39.7%）存在皮肤癣菌，而培养仅显示2例（7.40%）存在皮肤癣菌，临床敏感性提高了32.3%。在20份涂片阳性标本中，两种PCR-RFLP方法均在12份标本（17.6%）中检测到皮肤癣菌。两种方法在13份（19.11%）涂片和培养阴性标本中均检测到皮肤癣菌，临床敏感性提高了36.1%。
靶向真菌18S rDNA和ITS区域的PCR-RFLP方法对皮肤癣菌的检测和种属鉴定具有特异性和高度敏感性。
