1. rDNA基因序列分析法鉴定真菌菌种 2. 糠秕马拉色菌外分泌性蛋白分离及鉴定研究
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摘要
传统真菌菌种鉴定方法主要以表型特征为依据，即通过培养后观察菌落形态和显微特征，或辅以生理、生化、营养实验，但结果易受多种因素影响，或出现不能鉴定的情况。因此迫切需要建立一种稳定、可靠的真菌菌种鉴定方法。本研究包括以下五部分内容：
1．溶细胞酶结合Biospin真菌DNA提取试剂盒提取DNA
收集实验室保存菌株和临床分离真菌株，用溶细胞酶(lyticase)结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA，A_(260)／A_(280)检测纯度并计算质量浓度，经PCR扩增检验所提取的DNA质量，即采用真菌通用引物ITS1／ITS4扩增真菌核糖体RNA基因(ribosomal RNA gene, rDNA)内转录间区(internal transcribed spacers, ITS)。实验成功提取所有23株真菌基因组DNA，其纯度及质量浓度能满足PCR反应要求。用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒可以从酵母菌、无绿藻及丝状真菌提取DNA，所提取DNA可进一步用于PCR反应。为核糖体RNA基因序列扩增及序列分析奠定了基础。
2．草茎点霉的鉴定及超微结构研究
形态学结合核糖体RNA内转录间区(internal transcribed spacers, ITS)核酸序列分析的方法，对上级部门下发的1株质控菌株进行鉴定；组织病理切片、扫描电镜及透射电镜观察孢子器结构。表型研究显示：该菌在37℃可生长，40℃不能生长。在燕麦培养基中生长可见黑色点状菌落及灰白色绒毛状菌丝，后期见棕红色色素扩散至培养基中。镜下见有孔的球形或透镜形的黑色分生孢子器及器孢子，鉴定为茎点霉(Phoma)；核糖体RNA基因(rDNA)内转录间区ITS序列分析，证明与草茎点霉(Phoma herbarum, P.herbarum)ATCC 22167 ITS区一致性为100％，鉴定为草茎点霉。超微结构见孢子从分生孢子器内壁产生。该研究显示：形态学是菌种鉴定的基础，形态学结合ITS区测序的方法，不仅可使该菌株的鉴定达到种的水平，同时两种方法的鉴定结果互相验证，以保证鉴定结果准确、可靠。
3．祖菲无绿藻碳水化合物变种的鉴定及表型研究
根据形态学、生化特征，对1株分离自脑膜炎患者的无绿藻菌株进行菌种鉴定。该菌在25℃、40℃均可生长，沙堡弱培养基(SDA)、马铃薯培养基(PDA)见光滑湿润的白色酵母样菌落，镜下圆形、椭圆形厚壁孢子及裂殖成的圆形或不规则形内孢子。无菌丝、无子囊、无芽孢，鉴定为无绿藻。API 20C AUX鉴定系统提示与威克汉姆无绿藻一致性为2.9％。选用不同引物进行PCR反应，扩增产物纯化后直接测序，通过核酸序列数据库进行同源性序列搜索及一致性比较鉴定菌种。NL1／NL4引物所扩增核糖体大亚基(LSU)26S rDNA D1／D2序列，与祖菲无绿藻碳水化合物变种(Prototheca zopfii var. hydrocarbonea)一致性为96％，提示祖菲无绿藻碳水化合物变种的可能性大；核糖体内转录间区ITS序列可能存在复杂结构，不适合该菌的测序鉴定；真核细胞核糖体小亚基(SSU)18S rDNA通用引物及无绿藻种属特异性引物PCR产物，同源性序列搜索，证明分离株与祖菲无绿藻碳水化合物变种18S rDNA有99.9％的碱基同源序列，故最终鉴定为祖菲无绿藻碳水化合物变种。形态学是菌种鉴定的基础，形态学结合核酸序列分析的方法，可使该菌株的鉴定达到种的水平，在所选择的测序片段中，SSU 18S rDNA比LSU 26S rDNA序列分析，更有利于该临床分离株的鉴定。
4．真皮毛孢子菌的鉴定及生理、生化特征研究
采用沙堡弱培养基多点接种法，从1例29岁男性患者病甲中，分离出一株酵母样菌，做形态及API 20 C AUX鉴定，PCR反应扩增核糖体RNA基因(rDNA)内转录间区(ITS)、基因间区1(intergenic spacer 1，IGS1)分析后，用BLAST软件分析法进行菌种鉴定。半定量Api-Zym系统分析细胞外酶活性。该菌株在37℃生长良好，40℃不能生长，镜下为关节孢子和真、假菌丝。尿素酶阳性，API 20 C AUX结果与粘质隐球菌(Cryptococcus humicolus)一致率78.9％，尚不能鉴定至种的水平。ITS、IGS1序列与GenBank公布的真皮毛孢子ITS和IGS1区一致率分别为99.8％和100％，鉴定为真皮毛孢子菌(Trichosporon dermatis)。Api-Zym细胞外酶活性试剂盒分析，碱性磷酸酶、酯酶、类脂酯酶、类脂酶、白氨酸芳胺酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶、β-葡萄糖苷酶、N-乙酰-葡萄糖胺酶阳性。在形态学的基础上结合ITS、IGS1 rDNA序列分析可鉴定真皮毛孢子菌。多种酶活性可能与其致病有关。
5．从1例声带感染组织内分离鉴定烟曲霉
对一例曲霉所致的声带感染，进行包括直接镜检、培养、病理、镜下形态、温度耐受实验等真菌学研究及28S rDNA、ITS区测序等系列研究，将病原菌鉴定为烟曲霉(Aspergillus fumigatus)。
在真菌致病物质研究中，真菌分泌性蛋白的致病作用引起人们的关注。随着蛋白质分析技术的发展，蛋白质的分离及鉴定成为可能。该研究包括以下几个内容：①制备甘氨酸为唯一氮源培养基，糠秕马拉色菌在其中生长，甘油及油酸两种脂源可以促进生长。②收集培养上清，通过牛奶培养基分析法，分析上清酶活性，根据培养基成分被分解而形成的透明带，推测培养上清具有一定蛋白酶或类似蛋白酶活性。③通过Api-Zym酶活性分析，显示不同培养条件下，细胞外酶类型有一定差异。其中生理盐水菌悬液检测出6种酶活性，包括不同种类的脂酶、白氨酸芳胺酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶活性。在甘氨酸培养条件下，检测出类脂酯酶、酸性磷酸酶活性。在不同培养条件下，均未检测出胰蛋白酶、胰凝乳蛋白酶活性。④用Quanticleave蛋白酶分析法，检测到改良甘氨酸培养上清具有一定蛋白酶活性。加入BSA的改良甘氨酸培养上清，蛋白酶含量升高，提示培养基中的蛋白成分可诱导蛋白酶产生。⑤改良甘氨酸培养上清TCA沉淀后，经SDS-聚丙酰胺电泳进行蛋白初步分离，显示5条带，其中30 KD左右有两条带，丰度高。生理盐水菌悬液经超滤浓缩，SDS-聚丙酰胺电泳进行蛋白初步分离，显示5条带。但某些条带的分子量与改良甘氨酸上清来源的条带不同。
The traditional laboratory identification of fungi species is based on phenotypic methods, which are to investigate macroscopic colonial and microscopic morphology, or supplement with the physiological, biochemical characteristics and nutrition test. But the results often were interfered by diversiform factors, even the strain can not be judged. Therefore, the accurate and reliable identification of fungi to the level has become especially important.
1. Use of lyticase combined with Biospin Fungus Genomic DNA Extraction Kit to extract the DNA
Lyticase combined with Biopin Fungus Genomic DNA Extraction Kit were used to extract the fungal DNA from 23 fungus strains including clinical isolated and stored strains, according to the manufacturer's protocol. The DNA purity was checked by analyzing the ratio of A_(260)/A_(280) and its concentration was calculated. The universal fungal primers ITS1/ITS4 were used for PCR reaction, to amplify internal transcribed spacers (ITS) region of ribosome RNA gene (rDNA), using the gained DNA as the templates. All of 23 strains tested fungal genomic DNA could be extracted successfully, the DNA purity and concentration can meet with the need of PCR reactions. It showed that combination of lyticase with Biospin Fungus Genomic DNA Extraction Kit is a convenient and feasible way to extract genomic DNA from common clinical yeasts and filamentous fungi for PCR reaction. It will provide groundwork for the amplification of ribosomal RNA gene and sequence analysis.
2. Identification of Phoma herbarum and the study of its ultrastructure.
Morphologic methods and internal transcribed spacers (ITS) sequence analysis were used to identify a query strain fungus for quality control from higher authorities and its structure was observed by histopathological section, scanning and transmission electron microscope. The query strain could grow well at 37℃, not 40℃. Dark colonies and grey-white fluffy mycelium developed in oatmeal agar (OA) medium. A brown-reddish pigment was exuded into agar in the late stage of growth. Microscopically, the fungus produced spherical or lens-shaped dark ostiolated pycnidia, and pyendiospores. The query strain was identified to be Phoma species. DNA sequence on ITS region of ribosome RNA gene (rDNA) from the query was 100% homology to the type strain Phom herbarum strain ATCC 22167, so it was identified to be Phoma herbarum. The conidia are produced from the inner wall of pycnidia. It suggested that morphologic characteristic was the base for identifying Phoma herbarium. Combined with sequence analysis of ITS, the query fungus strain could be identified to the species level. The results could be validated each other, with more reliable and accurate.
3. Identification of Prototheca zopfii var. hydrocarbonea and the study of phenotype
The identification of a strain Prototheca isolated from a systematic protothecosis, was operated by routine morphologic and biochemical methods, and amplified the region of large-subunit (LSU) ribosomal RNA gene and intergenic space (ITS) region with universal fungal primers, with eukaryote-specific primers and Prototheca genus specific primer for the region of small-subunit (SSU) ribosomal RNA gene, compare the divergence in sequence alignment with the maximal-likehood methods for recognizing the query strain. It could grow well at 25℃and 40℃, smooth white colony, globular or ovoid cell, in which a number of round, ovoid unicellular spores with thick cell wall, no hypha, ascus and blastic conidia could be seen by culturing in Sabouraud dextrose agar. Based on the microbiological, the pathogen was confirmed as Prototheca, and 2.9% identities with Prototheca wickerhamii according to API 20C AUX system. Sequence chart showed that ITS region may be a complex structure, not suitable for the identification of Prototheca, and 99% sequence identities in the SSU region, 94% in the LSU region. It suggested that SSU rDNA sequence may be more reliable than LSU for the identification of this strain of Prototheca zopfii var. hydrocarbonea. It showed that microscopic and macroscopic characteristics are the base for identifying Prototheca zopfii. Analysis of nucleotide sequence can identify the query strain to the species level, among these choice, it could be seen that the sequence of SSU rDNA region may be more reliable than LSU for identifying this strain of Prototheca.
4. Identification of Trichosporon dermatis and the study of physiological, biochemical characteristics
To report a strain of Trichosporon dermatis isolated from onychomycosis and its extracellular enzymatic activities. The patient was a 29-year-old male with onychomycosis on his fingernails for more than 1 year. Yeast colonies were isolated from the nail scraps through multi-point inoculation on Sabouraud dextrose agar. Further identifications were performed on morphology, API 20C AUX identification system, PCR was used to amplify the internal transcribed spacer (ITS) and intergenic spacer 1 (IGS1) regions of ribosome DNA (rDNA). Then, compared the sequences with the ones posted on BLAST. The extracellular enzymatic activities were analysed with the semiquantitative Api-Zym system (bioMerieux sa, France). The strain grew well at 37℃, but not at 40℃, characterized by the arthroconidia, hyphae and pseudohyphae, with positive urease response. It can not be identified as Cryptococcus humicolus with 78.9% of the identity, determined by API 20C AUX identification system. Sequence analysis of the ITS and IGS1 regions were 99.8% and 100% identity to the ones of Trichosporon dermatis posted on BLAST. Therefore, it was identified to be Trichosporon dermatis. The strain expressed the activities of alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,β-glusidase, and acetyl-β-gluco-saminidase, detected by Api-Zym system. This was the first case that Trichosporon dermatis was isolated from onychomychosis in China. In addition to morphology, the pathogen was identified by nucleotide sequence analysis. Its multiple enzymatic activities may play a key role for pathogenesis of onychomycosis.
5. Isolate and identify a strain of Aspergillus fumigatus from infected vocal cord tissue
To identify the pathogen of a vocal cord inflammation, some investigations were operated, including directly microscopic examination by the KOH preparation, culture, pathology, macroscopic colonial morphology, temperature tolerance test and the sequence analysis of 28S rDNA and ITS. The microorganism was recognized to be Aspergillus fumigatus.
The secreted protein plays more roles in the pathogenesis of medical fungi. With the development of proteomic analysis, isolation and identification of protein become possible.①For protein analysis, Malassezia furfur grew in the only nitrogen source of glycine, and additional glycerin and oleic acid can promote the growth.②Collected the culture supernatants to analysis extracellular enzyme by fat-milk plate. The supernatants can degrade the fat-milk via extracellular enzymes system. It suggested that some protease activity or analogous protease was secreted and detected in culture supernatants.③Different culture can induce different extracellular enzymes activity by analysis of Api-Zym system. The secreted enzymes of Malassezia furfur are most productive in saline organism's suspension, including diverse esterase, leucine arylmidase, naphthol-AS-BI-phosp-hohy-drolase. Esterase lipase and esterase was induced in glycine medium. No trypsine and chymotrypsine was detected in diverse medium.④Protease activity was detected by the method of Quanticleave protease assay, and protease activity was higer in the glycine medium with BSA. It suggested that the protein in culture can induce protease.⑤Protein sample from glycine culture was precipitated with TCA and analyzed by SDS-PAGE. Five bands were displayed in SDS-PAGE gels and two proteins were abundant for about 30KD of molecular weight. 0.9% sodium chloride organism's suspension was filtrated, concentrated, isolated by SDS-PAGE. Five bands were displayed in SDS-PAGE, but some bands of molecular weight are different from protein sample from glycine culture.
1．溶细胞酶结合Biospin真菌DNA提取试剂盒提取DNA
收集实验室保存菌株和临床分离真菌株，用溶细胞酶(lyticase)结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA，A_(260)／A_(280)检测纯度并计算质量浓度，经PCR扩增检验所提取的DNA质量，即采用真菌通用引物ITS1／ITS4扩增真菌核糖体RNA基因(ribosomal RNA gene, rDNA)内转录间区(internal transcribed spacers, ITS)。实验成功提取所有23株真菌基因组DNA，其纯度及质量浓度能满足PCR反应要求。用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒可以从酵母菌、无绿藻及丝状真菌提取DNA，所提取DNA可进一步用于PCR反应。为核糖体RNA基因序列扩增及序列分析奠定了基础。
2．草茎点霉的鉴定及超微结构研究
形态学结合核糖体RNA内转录间区(internal transcribed spacers, ITS)核酸序列分析的方法，对上级部门下发的1株质控菌株进行鉴定；组织病理切片、扫描电镜及透射电镜观察孢子器结构。表型研究显示：该菌在37℃可生长，40℃不能生长。在燕麦培养基中生长可见黑色点状菌落及灰白色绒毛状菌丝，后期见棕红色色素扩散至培养基中。镜下见有孔的球形或透镜形的黑色分生孢子器及器孢子，鉴定为茎点霉(Phoma)；核糖体RNA基因(rDNA)内转录间区ITS序列分析，证明与草茎点霉(Phoma herbarum, P.herbarum)ATCC 22167 ITS区一致性为100％，鉴定为草茎点霉。超微结构见孢子从分生孢子器内壁产生。该研究显示：形态学是菌种鉴定的基础，形态学结合ITS区测序的方法，不仅可使该菌株的鉴定达到种的水平，同时两种方法的鉴定结果互相验证，以保证鉴定结果准确、可靠。
3．祖菲无绿藻碳水化合物变种的鉴定及表型研究
根据形态学、生化特征，对1株分离自脑膜炎患者的无绿藻菌株进行菌种鉴定。该菌在25℃、40℃均可生长，沙堡弱培养基(SDA)、马铃薯培养基(PDA)见光滑湿润的白色酵母样菌落，镜下圆形、椭圆形厚壁孢子及裂殖成的圆形或不规则形内孢子。无菌丝、无子囊、无芽孢，鉴定为无绿藻。API 20C AUX鉴定系统提示与威克汉姆无绿藻一致性为2.9％。选用不同引物进行PCR反应，扩增产物纯化后直接测序，通过核酸序列数据库进行同源性序列搜索及一致性比较鉴定菌种。NL1／NL4引物所扩增核糖体大亚基(LSU)26S rDNA D1／D2序列，与祖菲无绿藻碳水化合物变种(Prototheca zopfii var. hydrocarbonea)一致性为96％，提示祖菲无绿藻碳水化合物变种的可能性大；核糖体内转录间区ITS序列可能存在复杂结构，不适合该菌的测序鉴定；真核细胞核糖体小亚基(SSU)18S rDNA通用引物及无绿藻种属特异性引物PCR产物，同源性序列搜索，证明分离株与祖菲无绿藻碳水化合物变种18S rDNA有99.9％的碱基同源序列，故最终鉴定为祖菲无绿藻碳水化合物变种。形态学是菌种鉴定的基础，形态学结合核酸序列分析的方法，可使该菌株的鉴定达到种的水平，在所选择的测序片段中，SSU 18S rDNA比LSU 26S rDNA序列分析，更有利于该临床分离株的鉴定。
4．真皮毛孢子菌的鉴定及生理、生化特征研究
采用沙堡弱培养基多点接种法，从1例29岁男性患者病甲中，分离出一株酵母样菌，做形态及API 20 C AUX鉴定，PCR反应扩增核糖体RNA基因(rDNA)内转录间区(ITS)、基因间区1(intergenic spacer 1，IGS1)分析后，用BLAST软件分析法进行菌种鉴定。半定量Api-Zym系统分析细胞外酶活性。该菌株在37℃生长良好，40℃不能生长，镜下为关节孢子和真、假菌丝。尿素酶阳性，API 20 C AUX结果与粘质隐球菌(Cryptococcus humicolus)一致率78.9％，尚不能鉴定至种的水平。ITS、IGS1序列与GenBank公布的真皮毛孢子ITS和IGS1区一致率分别为99.8％和100％，鉴定为真皮毛孢子菌(Trichosporon dermatis)。Api-Zym细胞外酶活性试剂盒分析，碱性磷酸酶、酯酶、类脂酯酶、类脂酶、白氨酸芳胺酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶、β-葡萄糖苷酶、N-乙酰-葡萄糖胺酶阳性。在形态学的基础上结合ITS、IGS1 rDNA序列分析可鉴定真皮毛孢子菌。多种酶活性可能与其致病有关。
5．从1例声带感染组织内分离鉴定烟曲霉
对一例曲霉所致的声带感染，进行包括直接镜检、培养、病理、镜下形态、温度耐受实验等真菌学研究及28S rDNA、ITS区测序等系列研究，将病原菌鉴定为烟曲霉(Aspergillus fumigatus)。
在真菌致病物质研究中，真菌分泌性蛋白的致病作用引起人们的关注。随着蛋白质分析技术的发展，蛋白质的分离及鉴定成为可能。该研究包括以下几个内容：①制备甘氨酸为唯一氮源培养基，糠秕马拉色菌在其中生长，甘油及油酸两种脂源可以促进生长。②收集培养上清，通过牛奶培养基分析法，分析上清酶活性，根据培养基成分被分解而形成的透明带，推测培养上清具有一定蛋白酶或类似蛋白酶活性。③通过Api-Zym酶活性分析，显示不同培养条件下，细胞外酶类型有一定差异。其中生理盐水菌悬液检测出6种酶活性，包括不同种类的脂酶、白氨酸芳胺酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶活性。在甘氨酸培养条件下，检测出类脂酯酶、酸性磷酸酶活性。在不同培养条件下，均未检测出胰蛋白酶、胰凝乳蛋白酶活性。④用Quanticleave蛋白酶分析法，检测到改良甘氨酸培养上清具有一定蛋白酶活性。加入BSA的改良甘氨酸培养上清，蛋白酶含量升高，提示培养基中的蛋白成分可诱导蛋白酶产生。⑤改良甘氨酸培养上清TCA沉淀后，经SDS-聚丙酰胺电泳进行蛋白初步分离，显示5条带，其中30 KD左右有两条带，丰度高。生理盐水菌悬液经超滤浓缩，SDS-聚丙酰胺电泳进行蛋白初步分离，显示5条带。但某些条带的分子量与改良甘氨酸上清来源的条带不同。
The traditional laboratory identification of fungi species is based on phenotypic methods, which are to investigate macroscopic colonial and microscopic morphology, or supplement with the physiological, biochemical characteristics and nutrition test. But the results often were interfered by diversiform factors, even the strain can not be judged. Therefore, the accurate and reliable identification of fungi to the level has become especially important.
1. Use of lyticase combined with Biospin Fungus Genomic DNA Extraction Kit to extract the DNA
Lyticase combined with Biopin Fungus Genomic DNA Extraction Kit were used to extract the fungal DNA from 23 fungus strains including clinical isolated and stored strains, according to the manufacturer's protocol. The DNA purity was checked by analyzing the ratio of A_(260)/A_(280) and its concentration was calculated. The universal fungal primers ITS1/ITS4 were used for PCR reaction, to amplify internal transcribed spacers (ITS) region of ribosome RNA gene (rDNA), using the gained DNA as the templates. All of 23 strains tested fungal genomic DNA could be extracted successfully, the DNA purity and concentration can meet with the need of PCR reactions. It showed that combination of lyticase with Biospin Fungus Genomic DNA Extraction Kit is a convenient and feasible way to extract genomic DNA from common clinical yeasts and filamentous fungi for PCR reaction. It will provide groundwork for the amplification of ribosomal RNA gene and sequence analysis.
2. Identification of Phoma herbarum and the study of its ultrastructure.
Morphologic methods and internal transcribed spacers (ITS) sequence analysis were used to identify a query strain fungus for quality control from higher authorities and its structure was observed by histopathological section, scanning and transmission electron microscope. The query strain could grow well at 37℃, not 40℃. Dark colonies and grey-white fluffy mycelium developed in oatmeal agar (OA) medium. A brown-reddish pigment was exuded into agar in the late stage of growth. Microscopically, the fungus produced spherical or lens-shaped dark ostiolated pycnidia, and pyendiospores. The query strain was identified to be Phoma species. DNA sequence on ITS region of ribosome RNA gene (rDNA) from the query was 100% homology to the type strain Phom herbarum strain ATCC 22167, so it was identified to be Phoma herbarum. The conidia are produced from the inner wall of pycnidia. It suggested that morphologic characteristic was the base for identifying Phoma herbarium. Combined with sequence analysis of ITS, the query fungus strain could be identified to the species level. The results could be validated each other, with more reliable and accurate.
3. Identification of Prototheca zopfii var. hydrocarbonea and the study of phenotype
The identification of a strain Prototheca isolated from a systematic protothecosis, was operated by routine morphologic and biochemical methods, and amplified the region of large-subunit (LSU) ribosomal RNA gene and intergenic space (ITS) region with universal fungal primers, with eukaryote-specific primers and Prototheca genus specific primer for the region of small-subunit (SSU) ribosomal RNA gene, compare the divergence in sequence alignment with the maximal-likehood methods for recognizing the query strain. It could grow well at 25℃and 40℃, smooth white colony, globular or ovoid cell, in which a number of round, ovoid unicellular spores with thick cell wall, no hypha, ascus and blastic conidia could be seen by culturing in Sabouraud dextrose agar. Based on the microbiological, the pathogen was confirmed as Prototheca, and 2.9% identities with Prototheca wickerhamii according to API 20C AUX system. Sequence chart showed that ITS region may be a complex structure, not suitable for the identification of Prototheca, and 99% sequence identities in the SSU region, 94% in the LSU region. It suggested that SSU rDNA sequence may be more reliable than LSU for the identification of this strain of Prototheca zopfii var. hydrocarbonea. It showed that microscopic and macroscopic characteristics are the base for identifying Prototheca zopfii. Analysis of nucleotide sequence can identify the query strain to the species level, among these choice, it could be seen that the sequence of SSU rDNA region may be more reliable than LSU for identifying this strain of Prototheca.
4. Identification of Trichosporon dermatis and the study of physiological, biochemical characteristics
To report a strain of Trichosporon dermatis isolated from onychomycosis and its extracellular enzymatic activities. The patient was a 29-year-old male with onychomycosis on his fingernails for more than 1 year. Yeast colonies were isolated from the nail scraps through multi-point inoculation on Sabouraud dextrose agar. Further identifications were performed on morphology, API 20C AUX identification system, PCR was used to amplify the internal transcribed spacer (ITS) and intergenic spacer 1 (IGS1) regions of ribosome DNA (rDNA). Then, compared the sequences with the ones posted on BLAST. The extracellular enzymatic activities were analysed with the semiquantitative Api-Zym system (bioMerieux sa, France). The strain grew well at 37℃, but not at 40℃, characterized by the arthroconidia, hyphae and pseudohyphae, with positive urease response. It can not be identified as Cryptococcus humicolus with 78.9% of the identity, determined by API 20C AUX identification system. Sequence analysis of the ITS and IGS1 regions were 99.8% and 100% identity to the ones of Trichosporon dermatis posted on BLAST. Therefore, it was identified to be Trichosporon dermatis. The strain expressed the activities of alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase,β-glusidase, and acetyl-β-gluco-saminidase, detected by Api-Zym system. This was the first case that Trichosporon dermatis was isolated from onychomychosis in China. In addition to morphology, the pathogen was identified by nucleotide sequence analysis. Its multiple enzymatic activities may play a key role for pathogenesis of onychomycosis.
5. Isolate and identify a strain of Aspergillus fumigatus from infected vocal cord tissue
To identify the pathogen of a vocal cord inflammation, some investigations were operated, including directly microscopic examination by the KOH preparation, culture, pathology, macroscopic colonial morphology, temperature tolerance test and the sequence analysis of 28S rDNA and ITS. The microorganism was recognized to be Aspergillus fumigatus.
The secreted protein plays more roles in the pathogenesis of medical fungi. With the development of proteomic analysis, isolation and identification of protein become possible.①For protein analysis, Malassezia furfur grew in the only nitrogen source of glycine, and additional glycerin and oleic acid can promote the growth.②Collected the culture supernatants to analysis extracellular enzyme by fat-milk plate. The supernatants can degrade the fat-milk via extracellular enzymes system. It suggested that some protease activity or analogous protease was secreted and detected in culture supernatants.③Different culture can induce different extracellular enzymes activity by analysis of Api-Zym system. The secreted enzymes of Malassezia furfur are most productive in saline organism's suspension, including diverse esterase, leucine arylmidase, naphthol-AS-BI-phosp-hohy-drolase. Esterase lipase and esterase was induced in glycine medium. No trypsine and chymotrypsine was detected in diverse medium.④Protease activity was detected by the method of Quanticleave protease assay, and protease activity was higer in the glycine medium with BSA. It suggested that the protein in culture can induce protease.⑤Protein sample from glycine culture was precipitated with TCA and analyzed by SDS-PAGE. Five bands were displayed in SDS-PAGE gels and two proteins were abundant for about 30KD of molecular weight. 0.9% sodium chloride organism's suspension was filtrated, concentrated, isolated by SDS-PAGE. Five bands were displayed in SDS-PAGE, but some bands of molecular weight are different from protein sample from glycine culture.
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