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研究生:曾楷珺
研究生(外文):Tzeng, Kai-Jiun
論文名稱:環境來源之棘阿米巴原蟲及退伍軍人桿菌其變性梯度凝膠電泳鑑別技術及抗生素添加對棘阿米巴原蟲生長影響之研究
論文名稱(外文):Identification of Environmental Acanthamoeba and Legionella by Denaturing Gradient Gel Electrophoresis and the Influence of Antibiotics Addition for Acanthamoeba Cultivation
指導教授:許昺慕
指導教授(外文):Hsu, Bing-Mu
口試委員:胥直利林威辰吳淑芬陳建易
口試委員(外文):Hsu, Chih-LiLin, Wei-ChenWu, Shu-FenChen, Chien-Yen
口試日期:2014-06-16
學位類別:碩士
校院名稱:國立中正大學
系所名稱:應用地球物理研究所
學門:自然科學學門
學類:地球科學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:131
中文關鍵詞:棘阿米巴原蟲奈氏阿米巴原蟲哈氏阿米巴原蟲嗜肺性退伍軍人桿菌即時定量聚合酵素鏈鎖反應變性梯度凝膠電泳
外文關鍵詞:AcanthamoebaNaegleriaHartmanellaLegionella pneumophilareal-time PCRDGGE
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棘阿米巴原蟲、奈氏阿米巴原蟲與哈氏阿米巴原蟲皆屬於自由型阿米巴原蟲,它們廣泛的存在於各種環境水體中。有些阿米巴物種會對動物與人類造成疾病,並作為退伍軍人桿菌的宿主,提高感染退伍軍人病的風險。本研究目的為探討台灣地區環境水體中自由型阿米巴原蟲與退伍軍人桿菌之分佈情形,並利用即時定量聚合酵素鏈鎖反應(Real-time qPCR)來做定量分析。從環境中分離之嗜肺性退伍軍人桿菌則是利用親緣演化分析(Phylogenetic analysis)與多位基因座序列分析法(MLST)分析進行分型比較。除此之外利用變性梯度凝膠電泳(DGGE)技術,分離一般電泳無法分離之棘阿米巴原蟲物種,建立一套快速辨認菌種類型的圖譜系統,之後比較環境水體中微生物的菌相及探討環境水體中微生物的多樣性。而環境中培養出來的自由型阿米巴原蟲,由於存在許多雜菌間接影響自由型阿米巴原蟲的生長,使用Amphotericin B、Gentamycin及Penicillin-Streptomycin測試抑菌抗生素劑量,並利用鏡檢觀察尋找自由型阿米巴原蟲最佳的生長條件。另外針對棘阿米巴原蟲本身對抗生素的耐受性,以瞭解抗生素對其之影響。
本研究於台灣主要溪流、水庫、溫泉區及朴子溪採集共395個樣本,並利用各種分析法檢測棘阿米巴原蟲、奈氏阿米巴原蟲、哈氏阿米巴原蟲與退伍軍人桿菌。定序分析結果顯示,在各類水體中Acanthamoeba主要檢出種別為T4基因型。奈氏阿米巴原蟲檢出菌種以Naegleria spp. 及Naegleria australiensis為主。哈氏阿米巴原蟲是以Hartmanella vermiformis為主,此種最常作為退伍軍人桿菌之宿主。Real-time qPCR定量結果顯示,環境水中棘阿米巴原蟲與退伍軍人桿菌之濃度分別介於4.2×102~2.4×105 copies/L與2.1×102~4.4×1010 copies/L。從環境分離之2株嗜肺性退伍軍人桿菌(Legionella pneumophila)經分子分型法分析。結果顯示,利用mip、rpoB和dotA三段特殊目標基因共同進行親緣演化分析,以MLST分析其型別, flaA, pilE, asd, mip, mompS, proA和neuA序列編號為(6, 10, 15, 28, 21, 14, 11)及(6, 10, 15, 28, 17, 14, 11);利用Latex之結果此2株嗜肺性退伍軍人桿菌皆為血清2~14型。
而利用變性梯度凝膠電泳辨認菌種類型的圖譜系統發現每種基因型都有獨特的圖譜,並且利用Totallab軟體分析,為圖譜定義數值,以數值來作為圖譜認定的佐證。利用抗生素進行阿米巴純化培養過程中,培養液會隨著抗生素增加而越來越清澈,而抑制棘阿米巴原蟲部分,Amphotericin B之最小抑菌濃度為7.8 μg/mL、Gentamycin之最小抑菌濃度為15.6 U/mL及Penicillin-Streptomycin之最小抑菌濃度為625 U/mL。本研究發展一套Acanthamoeba變性梯度凝膠電泳辨認菌種類型的圖譜系統,待技術成熟,希望在未來可助於病源菌之快速偵測及防治。

Acanthamoeba,Naegleria and Hartmanella are free-living amoebae. They are widely present in various environmental water. Some amoeba species of animals and humans will cause disease, and as Legionella host, they will raising the risk of infection Legionella's disease. The purpose of this study was to investigate the distribution of water environment in Taiwan free-type amoeba and Legionella species, and the use of real-time quantitative polymerase chain reaction (Real-time qPCR) to quantitative analysis. L. pneumophila separation from the environment is the use of genetic evolution analysis (Phylogenetic analysis) with multilocus sequence typing (MLST) analysis type comparison. In addition to using denaturing gradient gel electrophoresis (DGGE) technique, separation Acanthamoeba species can not be separated from the general electrophoresis, the establishment of a rapid mapping system to identify the type of bacteria, microorganisms in environmental water after comparing microflora and explore the microbial diversity in environmental water. The environment culeured free-living amoeba, bacteria indirect effect due to the presence of many free type amoeba growth, the use of Amphotericin B, Gentamycin and Penicillin-Streptomycin test bacteriostatic antibiotic dose and use microscopy looking for free type amoeba observed optimal growth conditions. And Acanthamoeba itself against antibiotic resistance, in order to understand the impact of their antibiotic.
This study in Taiwan's main streams, reservoirs, spa area and a total of 395 samples collected Putsu River, and use a variety of analytical methods to detect Acanthamoeba,Naegleria and Hartmanella. Sequence analysis showed that in all types of water Acanthamoeba species not detected for T4 major genotypes. Naegleria strains detected in Naegleria spp. And Naegleria australiensis based. Hartmanella is Hartmanella vermiformis based, such as host most Legionella species. Real-time qPCR quantitative results show that the concentration of environmental water Acanthamoeba and Legionella species ranged from 4.2×102~2.4×105 copies/L and 2.1×102~4.4×1010 copies/L. Analysis from the separation of the environment 2 Legionella pneumophila by molecular typing method. The results showed that the use of mip, rpoB and dotA three sections specific target genes joint phylogenetic analysis to MLST analysis of its type, flaA, pilE, asd, mip, mompS, proA and neuA sequence number (6, 10, 15, 28, 21, 14, 11) and (6, 10, 15, 28, 17, 14, 11); Latex use the results of this two L. pneumophila serum from 2 to 14 are all type.
Using denaturing gradient gel electrophoresis to identify the type of bacteria found in each genotype map system has unique patterns, and use Totallab software analysis, numerical definition for map to map the value identified as evidence. The use of antibiotics amoeba pure culture process, the culture medium with antibiotics will increase more and more clear, and inhibit Acanthamoeba part, the minimum inhibitory concentration of Amphotericin B was 7.8 μg / mL, the minimum Gentamycin inhibitory concentration of 15.6 U / mL and the minimum inhibitory concentration of Penicillin-Streptomycin was 625 U / mL. This study developed a Acanthamoeba strains denaturing gradient gel electrophoresis to identify the type of mapping system until the technology is mature, I hope in the future to help fast detection and prevention of pathogenic bacteria.

目錄
誌謝 ii
中文摘要 iii
Abstract v
目錄 vii
圖目錄 xi
表目錄 xii
第一章 前言 1
1.1 研究背景: 1
1.2 研究目的 2
1.3 研究架構 3
第二章 文獻回顧 5
2.1 環境水體中致病微生物之微生物學、感染風險與感染案例 5
2.1.1 棘阿米巴原蟲 5
2.1.2 奈氏阿米巴原蟲 7
2.1.3 哈氏阿米巴原蟲 10
2.1.4退伍軍人桿菌 11
2.1.5退伍軍人桿菌在自由型阿米巴原蟲體內寄生之情形 12
2.2檢測方法 14
2.2.1自由型阿米巴原蟲檢測方法 14
2.2.2退伍軍人桿菌之培養基與檢測方法 15
2.3 聚合酶連鎖反應 19
2.4親緣關係分析與基因相似度分析 24
2.5 分子分型技術 25
2.6 變性梯度凝膠電泳 29
2.7 分子克隆 30
2.8 阿米巴原蟲抗生素實驗 31
第三章 研究設計 33
3.1 研究流程 33
3.2 環境水體樣本採集及處理 35
3.2.1環境水體樣本採集之地點 35
3.2.2 環境水體採樣所需設備及注意事項 38
3.2.3 環境水體樣本採集 40
3.2.4 採集環境水體樣本之濃縮 41
3.3 研究試劑及設備 41
3.3.1 研究試劑 41
3.3.2 研究設備 43
3.4 指標性微生物與物理性指標之檢測 45
3.4.1 指標性微生物檢測 45
3.4.2 物理性指標檢測 45
3.5 研究實驗之正控制組來源、保存方式與活化方法 46
3.5.1 大腸桿菌之正控制組來源 46
3.5.2 大腸桿菌之保存方式與活化方法 46
3.5.3 自由型阿米巴原蟲與退伍軍人桿菌之正控制組來源 47
3.5.4自由型阿米巴原蟲之保存與活化 47
3.5.5 退伍軍人桿菌之保存與活化 47
3.6 分子生物分析法 48
3.6.1 DNA萃取 48
3.6.2 引子設計 48
3.6.3 聚合酵素連鎖反應原理 49
3.6.4 PCR產物之檢測 49
3.7 自由型阿米巴原蟲之檢測方法 51
3.7.1 實驗流程 51
3.7.2 自由型阿米巴原蟲之培養法 52
3.7.3 自由型阿米巴原蟲之聚合酵素鏈鎖反應 54
3.8 退伍軍人桿菌檢測方法 57
3.8.1 實驗流程 57
3.8.2 退伍軍人桿菌之培養方法 58
3.8.3 退伍軍人桿菌之聚合酵素鏈鎖反應 60
3.8.4 嗜肺性退伍軍人桿菌之聚合酵素鏈鎖反應 61
3.8.5 嗜肺性退伍軍人桿菌之歐洲系統分型 62
3.8.6 直接螢光免疫抗體 64
3.9 變性梯度凝膠電泳 64
3.9.1 GC-clamp 64
3.9.2 DGGE配製 66
3.10 Clone 68
3.10.1 LB Agar配製: 68
3.10.2 Competent cell備製 68
3.10.3 核酸接合反應 69
3.10.4 Competent cell 轉型 69
3.11 自由型阿米巴原蟲抗生素實驗 70
3.12 統計之差異性分析 74
3.13 親緣關係分析與基因相似度分析 74
第四章 結果與討論 75
4.1 DGGE 75
4.1.1變性梯度凝膠電泳之Acanthamoeba標準菌種圖譜分析 75
4.1.2 DGGE圖譜之加成性 78
4.1.3 環境水樣之圖譜分析 81
4.1.4 環境水樣直接濃縮法與培養法之比較 86
4.2 退伍軍人桿菌DGGE菌群分析 88
4.2.1嗜肺性退伍軍人桿菌標準菌株及環境分離菌株之圖譜比較 88
4.2.2定序雜亂之退伍軍人桿菌環境樣本圖譜分析 90
4.3環境水體中阿米巴原蟲之抗藥性測試 92
4.3.1阿米巴培養法之抗生素抑制環境雜菌實驗 92
4.3.2阿米巴原蟲抗生素抗藥性濃度測試 95
4.4 環境水體調查 97
4.4.1溪流水體檢出情形 97
4.4.2 溪流水體之親緣演化分析 98
4.4.3 水庫水體檢出情形 102
4.4.4 水庫水體之親緣演化分析 104
4.4.5 溫泉水體檢出情形 107
4.4.6 溫泉水體之親緣演化分析 108
4.4.7 環境水體之水質指標差異性分析 110
4.4.7.1溪流水體之差異性分析 110
4.4.7.2 水庫水體之差異性分析 112
4.4.7.3 溫泉水體之差異性分析 112
4.5自由型阿米巴原蟲與退伍軍人桿菌之定量分析 113
4.5.1棘阿米巴原蟲定量分析 113
4.5.2 退伍軍人桿菌定量分析 116
4.6 嗜肺性退伍軍人桿菌分子分型法 119
第五章 結論 121
參考文獻 123






























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