【作者】 张晓利；

【导师】 刘维达； 李岷； 吕桂霞； 崔盘根； 龚向东；

【作者基本信息】 中国协和医科大学 ， 皮肤性病学， 2010， 博士

【摘要】 第一部分丝状真菌DNA提取方法的比较和优化目的筛选适合提取丝状真菌模板DNA的方法。方法比较2个菌落培养时间段(3d内和7-14d)提取DNA质量的差异；运用氯化苄法、CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量；选出最优方法,用8种常见丝状真菌进行检验和验证。结果培养3天内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验；4种方法制备的DNA均可用于PCR等后续实验,其中以CTAB法提取的DNA纯度好,产率最高。结论CTAB法是一种适用于丝状真菌DNA提取的简便方法。第二部分PCR-RFLP技术快速鉴别8种致病丝状真菌病原菌的实验研究目的建立能应用于临床检测深部丝状真菌感染病原菌的PCR-RFLP方法。方法用真菌通用引物扩增烟曲霉、黄曲霉、土曲霉、黑曲霉、杂色曲霉、构巢曲霉、尖端赛多孢和串珠镰刀菌的ITS区,分别用HhaⅠ、HaeⅢ、HinfⅠ、TaqⅠ和MspⅠ5种限制性核酸内切酶对PCR产物进行酶切,建立以PCR为基础的RFLP方法,然后对22株临床株和2株环境分离株进行PCR-RFLP图谱分析。结果对PCR产物进行RFLP分析可以准确鉴定8种深部致病丝状真菌,从DNA提取到酶切分析可以在一个工作日中完成；22株临床株和2株环境分离株PCR-RFLP鉴定结果与传统的形态学鉴定结果一致。结论PCR-RFLP技术是一种能够快速鉴定深部感染丝状真菌的有效方法。第三部分多重PCR检测深部曲霉感染病原菌的实验研究目的建立能同时鉴别多种曲霉病原菌的多重PCR体系。方法筛选4种最常见深部曲霉感染病原菌(烟曲霉、黄曲霉、土曲霉和黑曲霉)的特异性引物建立多重PCR体系,用该体系检测4种曲霉单模板、双模板和三模板的扩增情况,用DNA模板连续稀释的方式检测该反应体系的敏感性。然后对22株临床株和2株环境分离株进行检测。结果该多重PCR体系有较好的特异性,4种曲霉分别扩增出250 bp,200bp,450 bp和150 bp的DNA片段,电泳图中能够很好区别；在合适的反应条件下,对单模板、双模板和三模板均能扩增出目的片段,未见明显非特异片段的干扰；多重PCR体系的敏感性为100pg,较单种特异性引物的敏感性(10pg)略差。结论多重PCR体系能够检测4种曲霉的单纯感染和混合感染,是一种快速、敏感、特异性强的诊断方法。第四部分菌落PCR快速鉴别丝状真菌的实验研究目的探讨菌落PCR在检测丝状真菌病原菌方面的运用。方法初步建立丝状真菌菌落PCR的检测技术,用19种丝状真菌标准株进行验证,选取部分菌种的菌落PCR产物和酶切结果与常规PCR产物和酶切结果进行比较；所有菌落PCR产物进行测序,检测其准确性。结果19株菌中有16株(占84.2%)菌落PCR扩增成功；其中有7株菌进行了菌落PCR与常规PCR产物及酶切图谱的比较,结果条带完全一致；16株扩增成功的菌株ITS区测序结果与传统形态学结果一致。结论菌落PCR可以用于丝状真菌的快速检测,不仅缩短了菌落形态学鉴定所需的较长时间,而且也节省了制备模板DNA的时间和成本,是一种快速、经济的PCR检测技术。第五部分菌落PCR快速鉴定菌种的临床应用目的探讨菌落PCR在临床标本检测中的应用。方法收集1例皮下真菌感染的临床标本,在真菌培养长出菌落伊始即进行菌落PCR检测,扩增ITS区进行测序,鉴定病原菌的菌种；收集浅部真菌感染的临床标本(指趾甲、皮屑)11例,选择真菌通用引物NS、皮肤癣菌特异性引物和酵母菌特异性引物组成的多重PCR体系,用菌落PCR的方法进行扩增后,根据扩增片段大小判断病原菌的种属。结果菌落PCR鉴定皮下真菌感染病例的病原菌在取材后的第4天完成,鉴定菌种与传统形态学方法鉴定结果一致；菌落PCR鉴定浅部真菌感染病原菌在3-5d内完成,能将病原菌初步鉴定至种属水平,较传统形态学鉴定节省10d左右的时间。结论菌落PCR技术检测临床标本快速简便,适合临床实验室常规使用。更多还原

【Abstract】 Part I Comparison and optimization of DNA extraction method for filamentous fungiObjective To find the best DNA extraction methods for filamentous fungi. Methods We compared the DNA quality of fungi cultured for 3d and 7-14d. As a sample, Aspergillus. niger DNA was extracted by four methods, including benzyl chloride method, CTAB method, Biospin kit and microwave method, and compared the purity and output of the products via agarose gel electrophoresis and PCR. The best method was used to extract 8 pathogenic mould. Results The extracted DNA from colony cultured for 3d was purer than that for 7-14d. Comparing the DNA agarose gel electrophoretogram made by the four methods, all the DNA was amplifiable using a standard PCR and the DNA extracted by CTAB showed the best result. Conclusions CTAB is suit to extract mould DNA and has the advantages of high extraction rate, good quality and simple operation.Part II Experimental investigation on the identification for eight invasive pathogenic molds by PCR-RFLPObjective To establish a diagnostic method for invasive mould infections using PCR-RFLP. Methods The ITS regions of Aspergillus. fumigatus, Aspergillus. flavus, Aspergillus. Terreus, Aspergillus. niger, Aspergillus. Versicolor, Aspergillus. Nidulans, Scedosporium apiospermum and Fusarium moniliforme were amplified with universal fungal primers, and RFLP analysis with HhaⅠ, HinfⅠ,HaeⅢ, TaqⅠand MspⅠ. Then 22 clinical strains and 2 environmental isolates were analyzed with PCR-RFLP. Results RFLP analysis of the PCR products with Hha I and Hinf I allowed discrimination of 8 invasive pathogenic molds. DNA extraction, PCR and restriction digestion could be carried out within one day.22 clinical strains and 2 environmental isolates were completely identical to those obtained by conventional morphological methods. Conclusions PCR-RFLP is a rapid method for the identification of deep mold infection.PartⅢExperimental investigation on the detection for pathogens of invasive aspergillosis by multiplex PCR Objective To develop a multiplex PCR method to identify simultaneously multiple aspergillus pathogens in a single reaction. Methods Four pairs of primers were selected in one multiplex PCR to identify the most frequent Aspergillus pathogens, A. fumigatus, A. flavus, A. terreus and A. niger. Mutiplex PCR with these 4 primer pairs was used to detect 1 template and 2 or 3 template mixtures. Sensitivity of multiplex PCR was tested with serial dilutions of PCR products. A total of 24 fungal isolates were tested with this method. Results The detection system demonstrated high specificity, that 4 aspergillus were identified through distinct amplicons of 250 bp,200 bp,450 bp and150 bp, respectively. Multiplex PCR could amplify the corresponding 1,2 or 3 DNA fragments, and the sensitivity of multiplex PCR was 100pg, which was less sensitive to single-primer-set PCR(10pg). Conclusions Multiplex PCR might be a rapid, sensitive and specific method for the diagnosis of invasive aspergillosis.Part IV Experimental investigation on the detection of mould by colony PCRObjective To detect the application of colony PCR in the field of identification of pathogenic mould. Methods 19 mould strains were examined using direct colony PCR to amplify the ITS region without DNA extraction. We compared parts of the strains by electrophoresis and PCR-RFLP using colony PCR and standard PCR. All of the strains were verified by analysis of fungal ITS sequences. Results 16 of the 19 strains(84.2%) yielded positive results on colony PCR. The cleavage maps of colony PCR-RFLP coincided with the standard PCR, and sequence analysis of the 16 strains were similar to those obtained by conventional morphological methods. Conclusions Colony PCR is a rapid method for the identification of mould, which not only reduces DNA template preparation time before PCR from mould colonies, but also reduces the cost of PCR.Part V Clinical application of colony PCR in fast fungi identificationObjective To study the application of colony PCR in the detection of clinical specimens. Methods Clinical specimens from a boy with subcutaneous fungal infections were collected, and examined using direct colony PCR to amplify the ITS region. Skin and nail samples were taken from patients with superficial mycosis and detected by colony multiplex PCR based on 3 pairs of primers, universal fungus-specific primer, dermatophyte-specific primer and yeast-specific primer. Results Pathogens identification of subcutaneous fungal infections using colony PCR was similar to those obtained by conventional morphological methods, and completed in 5d. Use of colony PCR reduced the time for identification of superficial mycosis to 3-5d, which was considerably faster than morphology identification (another additional 10d). Conclusions Colony PCR is a rapid and convenient method for the detection of clinical specimens, and may be routinely used in clinical laboratory.更多还原