1. Identify The Fungi Species By Analyzing Ribosomal RNA Gene 2. Isolation And Identification Of Secreted Proteins From Malassezia Furfur

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 DNALyticase 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 A₂₆₀/A₂₈₀ 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 phenotypeThe 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 characteristicsTo 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 tissueTo 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.â‘ For protein analysis, Malassezia furfur grew in the only nitrogen source of glycine, and additional glycerin and oleic acid can promote the growth.â‘¡Collected 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.â‘¢Different 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.â‘£Protease 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.