浅部真菌病临床标本致病菌DNA提取方法的初步研究

目的初步建立直接从浅部真菌病临床标本中提取致病菌DNA的方法，为PCR技术快速诊断浅部真菌病及相关研究奠定基础。方法采用蛋白酶K消化法、煮沸法和冻融法处理直接镜检阳性的临床标本，以经典的酚-氯法抽提致病菌DNA，并用真菌通用引物ITS1进行PCR扩增检验。结果共收集标本61例，其中有44例PCR可扩增出特异性条带，甲屑的PCR检出率为81．0％，皮屑为69．2％；PER检测为阴性的17例中有7例标本量较少，有5例菌量较少。整个DNA提取过程约需3h。结论本研究初步建立的直接从浅部真菌病临床标本中提取致病菌DNA的方法能满足后续PCR反应的需要：DNA提取效率与标本量和菌量有关。

Preliminary Study on Extracting DNA of Pathogenic Fungi From Clinical Specimens of Superficial Fungal Infection

Objective We develop a method for extracting DNA of pathogenic fungi from dermal specimens directly, in order to apply PCR assay in diagnosis of superficial fungal infection. Methods A procedure combined pmteinase K digestion with boiling and freezing- thawing was used to deal with the clinical specimens, then genomic DNA was extracted by phenol- chloroform method. The efficiency of this procedure was estimated by PCR assay based on ITS1 primer. Results Of 61 clinical samples which were positive on microscopy, forty - four were positive on PCR, and the samples from skin scrapings and nail shavings accounted for 69.2% and 81.0%, respectively. In 17 PCR- negative cases, less amounts of specimens and a small quantity of hyphae were found in 7 and 5 cases.It takes about 3h for the whole procedure for DNA extraction. Conclusion This method for DNA extraction could be useful to apply PCR assay in diagnosis of superficial fungal infection. The amount of specimen and the quantity of hyphae might affect the efficiency of the method.