脂溢性皮炎患者和健康志愿者面部馬拉色菌菌種差異分析
發(fā)布時間：2019-04-21 10:23
【摘要】：背景：馬拉色菌是一種嗜脂酵母，是人類正常常駐菌，為了研究馬拉色菌與馬拉色菌毛囊炎、脂溢性皮炎、特異性皮炎等疾病的發(fā)病關(guān)系，因此快速準(zhǔn)確的鑒定馬拉色菌菌種顯得尤為重要。 目的：1）比較表型鑒定方法和序列測定分析方法的差別。2）以本地區(qū)脂溢性皮炎患者的臨床株為研究對象，以健康志愿者菌株為對照組，在首先獲得每個菌株ITS區(qū)堿基序列后，，采用分子系統(tǒng)學(xué)軟件對基因片段序列進(jìn)行分析，將菌株鑒定到種，結(jié)合這些菌株的形態(tài)學(xué)特征、生理生化鑒定結(jié)果，確定馬拉色菌菌種分類，從而揭示本地區(qū)脂溢性皮炎（Seborrheic Dermatitis，SD）患者及健康志愿者面部馬拉色菌菌種差異。 方法：本研究對124株臨床分離株分別進(jìn)行形態(tài)學(xué)、生化試驗、DNA序列分析等方法的試驗，通過表型鑒定方法與分子生物學(xué)鑒定方法相互驗證來更可靠的對所得臨床株分類。本研究所用到的形態(tài)學(xué)方法主要包括直接鏡檢、觀察培養(yǎng)菌落形態(tài)、培養(yǎng)后菌株鏡檢，生化試驗方法主要采用沙氏培養(yǎng)基生長試驗、吐溫試驗、七葉苷分解試驗、過氧化氫試驗等傳統(tǒng)試驗方法，同時應(yīng)用分子生物學(xué)方法中的DNA序列分析法來對臨床收集到的菌株進(jìn)行鑒定。本研究采用玻璃珠破壁、酚-氯仿-異戊醇抽提法提取基因組DNA，并應(yīng)用真菌通用引物ITS4、ITS5選擇性PCR擴(kuò)增真菌基因組核糖體DNA（rDNA）的ITS1-5.8S-ITS2區(qū)，并對該區(qū)進(jìn)行測序，應(yīng)用DNAMAN、Bioedit軟件對測序所得DNA堿基序列進(jìn)行編輯，與Genbank數(shù)據(jù)庫中的序列比對，以此鑒定馬拉色菌菌種。應(yīng)用統(tǒng)計軟件SPSS ll.5分析兩種疾病的菌種構(gòu)成，顯著性檢驗水平為α=0.05。 結(jié)果：DNA測序鑒定結(jié)果與表型鑒定結(jié)果存在一定差異，有89.52%（111/124）的菌株兩方法的鑒定結(jié)果是一致的。以測序鑒定結(jié)果為準(zhǔn)，脂溢性皮炎患者面部菌株糠秕馬拉色菌49株占76.56%，隨后是合軸馬拉色菌8株占12.5%，M.japonica6株占9.38%，球形馬拉色菌1株占1.56%；健康志愿者面部菌株糠秕馬拉色菌37株占61.67%，合軸馬拉色菌15株占25.00%，M.japonica4株占6.67%，鈍形馬拉色菌、球形馬拉色菌均為2株各占3.33%。 結(jié)論：本研究中脂溢性皮炎患者和健康志愿者面部菌群總體構(gòu)成比差異無統(tǒng)計學(xué)意義，兩組人群面部帶菌均以糠秕馬拉色菌為主，其次是合軸馬拉色菌。表型鑒定方法和DNA序列分析法得出的鑒定結(jié)果存在一定差異。DNA序列分析法能快速、準(zhǔn)確的鑒定馬拉色菌菌種，以便臨床醫(yī)生對病人疾病管理快速做出針對性的決策。
[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 偽 = 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R756.5
本文編號：2462104
[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 偽 = 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R756.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 崔凡;沈永年;徐宏斌;胡素泉;呂桂霞;陳偉;劉維達(dá);;馬拉色菌屬鑒定中吐溫試驗新方法的研究[J];實用醫(yī)院臨床雜志;2010年06期
本文編號：2462104
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