臺灣博碩士論文加值系統

中文摘要
本研究以伺機性病原酵母菌Candida tropicalis 為對象，以國內菌株為主體，配合國外菌株之比對，蒐集天然、人為干擾與臨床環境共169菌株，利用脈衝電場膠體電泳(Pulsed-field gel electrophories, PFGE)進行電泳核型(Electrophoretic karyotypes)初步分析，後續藉由隨機擴增多型性DNA (Random amplified polymorphism DNA, RAPD)與XYR1基因的分型作篩選，擇其中65株為代表，串聯MDR1、SAPT4、XYR1、ZWF1-α四段基因，以總長1705個核苷酸的序列，探討台灣C. tropicalis在臨床與環境上的族群結構與親緣地理。初步分析結果顯示：C. tropicalis可依核型差異區分為15群，各群續以RSD8為引子作隨機擴增多型性DNA分析篩選出99菌株，XYR1基因片段之序列比對結果得33型，依序列及樣品特異性擇其中65菌株作族群與親緣地理分析。族群整體之結果顯示：65個菌株共可區分為60種基因單型(Haplotype)，族群內的核苷酸多樣性(Nucleotide diversity, π)為0.0062±0.004，族群間的基因單型多樣性(Haplotype diversity, h)為0.998±0.003，族群整體未偏離中性檢測結果，且有持續擴張之傾向，但兩兩族群間的分化程度與地理距離並無顯著相關，此結果顯示本菌種擴散力極強。另分子變異分析(Analysis of molecular variance, AMOVA)顯示：遺傳變異主要發生於族群內部，藉由巢狀支系分析(Nested clade analysis)，可知本菌之種源來自環境，再擴散於人體。若藉GeoDis的分析可知C. tropicalis族群現今的族群結構，是經歷了鄰域擴張、地理限制導致分化，以及長距離拓殖並伴隨著後續的片斷化事件的結果。藥物敏感性試驗顯示：天然環境菌株在Fluconazole的耐受性表現有較人為干擾與臨床環境菌株低的趨勢。

Abstract
Candida tropicalis, a commensal and opportunisitic human fungal pathogen, is prevalent in clinical environment. In this study, a total of 169 C. tropicalis strains were collected from natural, anthropogenic, and clinical environment. First authenticated and preliminary typed by electrophoretic karyotyping with pulsed field gel electrophoresis (PFGE) and random amplified polymorphism DNA (RAPD) with the primer RSD8. At last, 65 strains were selected to stand for the C. tropicalis population, and a DNA sequence connected together by MDR1, SAPT4, XYR1 and ZWF1-α genes with a total length of 1705 bp were used to assess the phylogeography of C. tropicalis. There were 60 haplotypes generated from the 65 strains. The high nucleotide diversity (π=0.0062±0.004) and high haplotype diversity (h=0.998±0.003) indicated a large stable population with long evolutionary history. Genetic variation extent was not corresponded to the distance between two of populations. We guess it might be interfered by the high popupation expansion of C. tropicalis. Analysis of molecular variance (AMOVA) revealed significant genetic subdivision occurred within the population. The results of nested clade analysis suggest that C. tropicalis population originated from natural environment flowed to clinical environment as a pathogen. Forward analysis of Geodis suggested that the present population structure was a result of contiguous range expansion, restricted gene flow with isolation by distance, and long distance colonization. At the last, sensitiveity test shows that strains isolated from natural environment present lower MIC in fluconazole than that of strains from anthropogenic and clinical environment.

目 錄

目錄 I
表目錄 III
圖目錄 IV
摘要 V
Abstract VI
壹、 前言 1
貳、 文獻整理 2
一、C. tropicalis分類與特徵 2
二、C. tropicalis生態與分布 2
三、病原性與治療 2
(一) 感染性與病原性 2
(二) C. tropicalis之臨床藥物與治療 4
(三) 臨床菌株分型 4
四、微生物親緣地理 5
(一) 親緣地理簡介與研究發展 5
(二) 親緣地理分析指標與依據 5
(三) 親緣地理分析方法 7
(四) 微生物在親緣地理上的研究 7
參、 材料與方法 10
一、藥品 10
二、培養基 10
三、器材 10
四、儀器 10
五、引子 10
六、研究方法 10
(一) 菌株來源 10
(二) DNA萃取 18
(三) CHROMagar形態分型法 18
(四) 隨機擴增多型性DNA (Random amplified polymorphism DNA, RAPD)分型法 18
(五) 脈衝電場膠體電泳(Pulsed-field gel electrophories, PFGE ) 19
(六) 多位基因座序列分型法(Multi-locus sequence typing, MLST) 20
(七) 藥物敏感性試驗 20
(八) 生理生化試驗 21
(九) 資料分析 21
肆、 結果 26
一、菌株複核 26
(一) 念珠菌顯色培養基CHROMagarTM Candida 26
(二) GTG隨機擴增多型性電泳圖譜 26



二、形態與生理生化試驗 26
(一) 生理生化試驗 26
(二) 形態試驗 26
三、染色體電泳核型分析(Electrophoretic karyotypes) 26
四、隨機擴增多型性DNA (Random amplified polymorphism DNA, RAPD) 36
五、多位基因座序列分型法(Multi-locus sequence typing, MLST) 36
六、藥物敏感性試驗 45
七、族群結構與親緣地理分析 45
(一) C. tropicalis 之核苷酸鹼基組成分析 45
(二) 遺傳變異分析(Genetic variation analysis ) 45
(三) 親緣分析(Phylogenetic analysis) 57
(四) 族群結構分析(Genetic structure analysis) 62
伍、 討論 67
一、C. tropicalis族群之遺傳變異與親緣地理關係探討 67
二、C. tropicalis環境族群與臨床族群之親緣探討 69
陸、 結論 72
柒、 參考文獻 73
捌、 附錄 77
附錄一、實驗藥品清單 77
附錄二、培養基製備方式 78
附錄三、C. tropicalis菌株生理生化試驗結果 79

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