脂溢性皮炎患者和健康志愿者面部马拉色菌菌种差异分析

发布时间：2019-04-21 10:23

【摘要】：背景：马拉色菌是一种嗜脂酵母，是人类正常常驻菌，为了研究马拉色菌与马拉色菌毛囊炎、脂溢性皮炎、特异性皮炎等疾病的发病关系，因此快速准确的鉴定马拉色菌菌种显得尤为重要。目的：1）比较表型鉴定方法和序列测定分析方法的差别。2）以本地区脂溢性皮炎患者的临床株为研究对象，以健康志愿者菌株为对照组，在首先获得每个菌株ITS区碱基序列后，，采用分子系统学软件对基因片段序列进行分析，将菌株鉴定到种，结合这些菌株的形态学特征、生理生化鉴定结果，确定马拉色菌菌种分类，从而揭示本地区脂溢性皮炎（Seborrheic Dermatitis，SD）患者及健康志愿者面部马拉色菌菌种差异。方法：本研究对124株临床分离株分别进行形态学、生化试验、DNA序列分析等方法的试验，通过表型鉴定方法与分子生物学鉴定方法相互验证来更可靠的对所得临床株分类。本研究所用到的形态学方法主要包括直接镜检、观察培养菌落形态、培养后菌株镜检，生化试验方法主要采用沙氏培养基生长试验、吐温试验、七叶苷分解试验、过氧化氢试验等传统试验方法，同时应用分子生物学方法中的DNA序列分析法来对临床收集到的菌株进行鉴定。本研究采用玻璃珠破壁、酚-氯仿-异戊醇抽提法提取基因组DNA，并应用真菌通用引物ITS4、ITS5选择性PCR扩增真菌基因组核糖体DNA（rDNA）的ITS1-5.8S-ITS2区，并对该区进行测序，应用DNAMAN、Bioedit软件对测序所得DNA碱基序列进行编辑，与Genbank数据库中的序列比对，以此鉴定马拉色菌菌种。应用统计软件SPSS ll.5分析两种疾病的菌种构成，显著性检验水平为α=0.05。结果：DNA测序鉴定结果与表型鉴定结果存在一定差异，有89.52%（111/124）的菌株两方法的鉴定结果是一致的。以测序鉴定结果为准，脂溢性皮炎患者面部菌株糠秕马拉色菌49株占76.56%，随后是合轴马拉色菌8株占12.5%，M.japonica6株占9.38%，球形马拉色菌1株占1.56%；健康志愿者面部菌株糠秕马拉色菌37株占61.67%，合轴马拉色菌15株占25.00%，M.japonica4株占6.67%，钝形马拉色菌、球形马拉色菌均为2株各占3.33%。结论：本研究中脂溢性皮炎患者和健康志愿者面部菌群总体构成比差异无统计学意义，两组人群面部带菌均以糠秕马拉色菌为主，其次是合轴马拉色菌。表型鉴定方法和DNA序列分析法得出的鉴定结果存在一定差异。DNA序列分析法能快速、准确的鉴定马拉色菌菌种，以便临床医生对病人疾病管理快速做出针对性的决策。
[Abstract]:Background: Malassezia is a lipophilic yeast and a normal resident bacteria. In order to study the relationship between Malassezia and malassezia folliculitis, seborrheic dermatitis, specific dermatitis and other diseases, Therefore, rapid and accurate identification of Malassezia species is particularly important. Objective: 1) to compare the difference between phenotypic identification and sequencing. 2) to study the clinical strains of seborrheic dermatitis patients in this area and the healthy volunteers as the control group. After the base sequences of ITS region of each strain were obtained firstly, the sequence of gene fragment was analyzed by molecular phylogeny software, and the strains were identified to species, combined with the morphological characteristics, physiological and biochemical identification results of these strains. The taxonomy of Malassezia spp. was determined to reveal the difference of Malassezia species in face between patients with seborrheic dermatitis (Seborrheic Dermatitis,SD) and healthy volunteers. Methods: in this study, the morphological, biochemical and DNA sequences of 124 clinical isolates were tested, and the clinical strains were classified more reliably by the methods of phenotypic identification and molecular biological identification. The morphological methods used in the study include direct microscopic examination, observation of colony morphology, and microscopic examination of the cultured strains. Biochemical methods are mainly used in the growth test of Shashi medium, Tween test, and the decomposition test of aescin, and the results are as follows: (1) the morphological methods used in this study include: 1. The traditional test methods, such as hydrogen peroxide test and DNA sequence analysis in molecular biology, were used to identify the clinical collected strains. In this study, genomic DNA, was extracted by phenol-chloroform-isoamyl alcohol extraction and the ITS1-5.8S-ITS2 region of genomic ribosomal DNA (rDNA) was amplified by selective PCR with fungal universal primer ITS4,ITS5, using glass beads broken wall and phenol-chloroform-isoamyl alcohol extraction method. The DNA base sequence was edited by DNAMAN,Bioedit software and compared with the sequence in Genbank database to identify Malassezia spp. by sequencing the sequence of Marassezia spp. in the region and using the Genbank software to edit the nucleotide sequence of Marassezia spp. The statistical software SPSS ll.5 was used to analyze the bacterial composition of the two diseases. The level of significance test was 伪 = 0.05. Results: there were some differences between the results of DNA sequencing and phenotypic identification. 89.52% (111 脳 124) of the strains were identified by the two methods. According to the result of sequencing, 49 strains of Malassezia furfur in seborrheic dermatitis patients accounted for 76.56%, 8 strains of Malassezia melatoria accounted for 12.5%, 6 strains of M. japonica accounted for 9.38%, and 1 strain of Malassezia globosa accounted for 1.56%. In healthy volunteers, 37 strains of Malassezia furfur, 15 strains of Malassezia paraaxi, 6.67% of M. japonica, 3.33% of Malassezia globosa and 37 strains of Malassezia furfur, 4 strains of M. japonica and 3.33% of Malassezia globosa were respectively. Conclusion: there was no significant difference between seborrheic dermatitis patients and healthy volunteers in the overall composition ratio of facial flora. Malassezia furfurum was the main bacteria in the two groups, followed by Malassezia melasma. There are some differences between phenotypic identification methods and DNA sequence analysis methods, which can quickly and accurately identify Malassezia spp. so that clinicians can quickly make targeted decisions on patient disease management.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756.5

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