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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張靜文(Ching-Wen Chang) | |
dc.contributor.author | Chung-Long Kuo | en |
dc.contributor.author | 郭忠龍 | zh_TW |
dc.date.accessioned | 2021-05-16T16:28:08Z | - |
dc.date.available | 2018-03-04 | |
dc.date.available | 2021-05-16T16:28:08Z | - |
dc.date.copyright | 2013-03-04 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-01-17 | |
dc.identifier.citation | 呂聆文(2010). 以核酸染劑結合即時定量聚合酶連鎖反應定量活性Acathamoeba. 國立台灣大學公共衛生學院環境衛生研究所碩士論文
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6396 | - |
dc.description.abstract | 棘阿米巴原蟲(Acanthamoeba spp.)為自由型阿米巴原蟲(free-living amoeba, FLA)的一種,存在於土壤、空氣以及人工與自然水體當中。暴露於活性棘阿米巴原蟲可感染棘阿米巴角膜炎(Acanthamoeba keratitis ,AK)、棘阿米巴皮膚炎(Cutaneous acanthamebiasis)以及肉芽腫性阿米巴腦膜炎(Granulomatous amoebic encephalitis, GAE),目前在環境當中,學術研究多屬定性之棘阿米巴原蟲檢出報告,以致難以量化各式環境之暴露風險。本研究嘗試建立以ethidium monoazide (EMA)搭配即時定量聚合酶連鎖反應(real-time quantitative polymerase chain reaction, qPCR)方法,以定量土壤、空氣以及水體中活性棘阿米巴原蟲,並分別於洋蔥田(N=12)、蔬果/香草植物田(N=36)及水稻田(N=5)採集表層土壤,於洋蔥田中央進行空氣採樣(N=12),於冷卻水塔(N=6)、廢水處理場(N=3)、農田溝渠(N=6)、水稻田(N=6)以及生物實驗室洗眼站(N=4)採集水體,再以所建立之EMA-qPCR及qPCR方法定量樣本中活性與總棘阿米巴原蟲濃度。土壤採樣同時也記錄當時土壤結構(鬆軟、堅硬但有破壞、堅硬)以及植被覆蓋狀況(無植物、有植物),並於實驗室分析土壤的pH值、含水量及異營性細菌濃度,另於空氣採樣時同步監測空氣中溫度、相對濕度及風速,本研究將土壤、空氣環境因子與活性及總棘阿米巴原蟲濃度進行統計分析,以評估影響土壤與空氣中活性與總棘阿米巴原蟲濃度之顯著相關因子。
在開發定量活性棘阿米巴原蟲部分,本研究將未受熱與受熱棘阿米巴原蟲囊體分別以0、2.3、23 μg/mL EMA或PMA處理5 min,再以500W鹵素燈距離15 cm光照20 min後以qPCR定量,數據結果顯示,經2.3 μg/mL EMA或PMA處理後,並不影響未受熱原蟲囊體準確定量,且可使受熱原蟲囊體下降約3 log DNA quantity,另外EMA-qPCR與PMA-qPCR在定量未受熱與受熱原蟲囊體之DNA上,經魏克森等級和檢定(Wilcoxon rank sums test)顯示無統計顯著差異(P=1.00)。 在定量土壤中活性棘阿米巴原蟲部分,本研究將未受熱與受熱原蟲添加至1X-300X滅菌土壤稀釋液當中,並以0、2.3、23、46 μg/mL EMA處理5 min再以500W鹵素燈距離15 cm或LED燈(PhAST Blue)光照20 min後以qPCR定量,結果發現50X土壤稀釋液搭配2.3 μg/mL EMA處理不影響未受熱原蟲準確定量亦可使受熱原蟲下降約4 log DNA quantity,且鹵素燈與LED燈對於定量未受熱與受熱棘阿米巴原蟲之能力經魏克森等級和檢定無統計上顯著差異(P=0.53)。 洋蔥田表層土壤活性棘阿米巴原蟲平均濃度(3.92×104 cells/g dry wt.)顯著高於蔬果/香草植物田(1.58×104 cells/g dry wt.)及水稻田(6.57×103 cells/g dry wt.)。在總棘阿米巴原蟲方面,洋蔥田平均濃度(8.13×104 cells/g dry wt.)亦顯著高於蔬果/香草植物田(3.23×104 cells/g dry wt.)及水稻田(1.24×104 cells/g dry wt.)。 洋蔥田空氣中活性與總棘阿米巴原蟲平均濃度分別為1.30與3.38 cells/m3,而經魏克森等級和檢定後,採收期總原蟲濃度(4.73 cells/m3)大於採收後(0.42 cells/m3)。 廢水處理廠活性與總棘阿米巴原蟲濃度以曝氣池水最高,分別為6.33×105與6.71×105 cells/L;進水管處之活性與總原蟲濃度次之,分別為4.52×104與5.78×104 cells/L,放流管處之活性與總原蟲濃度最低(5.23×102與1.20×103 cells/L)。冷卻水塔中活性與總棘阿米巴原蟲濃度為3.02×102與4.13×102 cells/L。農田環境中,水稻田水樣之活性與總棘阿米巴原蟲平均濃度分別為2.67×104與3.86×104 cells/L,農田溝渠水樣則降低至2.16×103與4.33×103 cells/L。在實驗室洗眼站樣本則未檢出。 以斯皮爾曼等級相關(Spearman's rank correlation)分析表層土壤中活性與總棘阿米巴原蟲濃度與土壤環境因子(土壤pH值、土壤含水量、土壤異營性細菌濃度、土壤結構以及植被覆蓋)之相關性,發現pH值(5.41-7.60)與土壤表層活性及總棘阿米巴原蟲濃度成正相關(r=0.60, P<0.0001;r=0.43, P=0.001);土壤異營性細菌濃度(2.21×107-7.74×108 CFU/g dry wt.)也與活性及總原蟲濃度呈正相關(r=0.33, P=0.02;r=0.39, P=0.004);而土壤含水量(2.19-58.23%)與活性及總原蟲濃度呈現負相關(r=-0.4, P=0.003;r=-0.37, P=0.006)。 以多元線性逐步迴歸(multiple linear regression with stepwise procedure)分析表層土壤中活性與總棘阿米巴原蟲濃度之影響因子,顯示pH值(5.41-7.60)增加可影響活性與總棘阿米巴原蟲濃度(β=21322, P<0.0001和β=37409, P=0.007),而土壤異營性細菌濃度(2.21×107-7.74×108 CFU/g dry wt.)增加亦有同樣作用(β=0.000064, P=0.007和β=0.00017, P=0.001)。 以斯皮爾曼等級相關分析空氣中活性與總棘阿米巴原蟲濃度(0.11-3.61 與0.20-6.94 cells/m3)、土壤表層活性與總原蟲濃度及環境因子(土壤pH值、土壤含水量、土壤異營性細菌濃度、土壤結構以及植被覆蓋、空氣溫度、空氣相對溼度、空氣風速)之相關性時,發現僅空氣中總棘阿米巴原蟲濃度與土壤植被覆蓋呈現正相關(r=0.52, P=0.08),顯示農田表面有植被覆蓋時,空氣中總棘阿米巴原蟲濃度較高。 本研究開發活性棘阿米巴原蟲之定量監測方法,並將其應用在土壤、空氣、水體當中以得知棘阿米巴原蟲濃度,並嘗試利用統計方法從土壤及空氣環境因子中找出影響棘阿米巴原蟲濃度變化的因子,然而本研究是偵測Acanthamoeba spp.,包含了致病性及非致病性,需要有研究去說明致病性與非致病性棘阿米巴原蟲在不同環境中的比例,方能清楚知道人在環境當中的暴露風險為何。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-05-16T16:28:08Z (GMT). No. of bitstreams: 1 ntu-102-R99844016-1.pdf: 3593380 bytes, checksum: f974cf291b4b9c75cea6672663081257 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 致謝 i
摘要 ii Abstract v 目錄 viii 圖次 xiv 表次 xvii 第一章 前言 1 1.1 研究背景 1 1.2 文獻回顧 1 1.2.1 棘阿米巴原蟲(Acanthamoeba spp.) 1 1.2.2 棘阿米巴原蟲導致之疾病及與土壤之相關性 2 1.2.3 棘阿米巴原蟲於土壤中之流佈及其影響因子 6 1.2.4 棘阿米巴原蟲於空氣中之流佈及其影響因子 9 1.2.5 棘阿米巴原蟲於水體中之流佈 14 1.2.6 環境棘阿米巴原蟲之偵測與定量方法 15 第二章 研究目的 18 第三章 研究架構 19 第四章 材料與方法 22 4.1 微生物菌種 22 4.1.1 Acanthamoeba castellanii (ATCC30234) 22 4.1.1.1 滋養體 22 4.1.1.2 囊體 22 4.1.2 Escherichia coli (ATCC25922) 22 4.2 試劑與緩衝液 22 4.2.1 製備ATCC medium 712 22 4.2.2 製備Page’s Amoeba Saline 23 4.2.3 製備囊化培養液(Encyst medium) 24 4.2.4 製備TE buffer 24 4.2.5 製備Nutrient agar (NA) 24 4.2.6 製備R2A agar 25 4.2.7 製備Non-Nutrient agar (NNA) 25 4.2.8 製備Heat-killed E.coli NNA plate 25 4.3 方法開發 26 4.3.1 棘阿米巴原蟲囊體核酸染劑之選擇 26 4.3.1.1 置備已知濃度的未受熱與受熱棘阿米巴原蟲囊體懸浮液 26 4.3.1.2 使用Heat-killed E.coli NNA plate確認未受熱與受熱囊體之活性 26 4.3.1.3 EMA與PMA核酸染劑之配置 26 4.3.1.4 EMA與PMA核酸染劑處理 27 4.3.1.5 DNA萃取 27 4.3.1.6 Real-time quantitative PCR (qPCR) 27 4.3.1.7 製備棘阿米巴原蟲DNA標準品及檢量線 28 4.3.1.8 定量指標、ND之定義及處理 29 4.3.2 EMA-qPCR定量土壤中活性棘阿米巴原蟲之方法開發 30 4.3.2.1 未滅菌土壤添加未受熱與受熱棘阿米巴原蟲進行不同濃度EMA與鹵素燈處理 30 4.3.2.1.1 製備已知濃度的未受熱與受熱原蟲懸浮液 30 4.3.2.1.2 未受熱與受熱原蟲土壤混合液配置 30 4.3.2.1.3 核酸染劑處理 30 4.3.2.1.4 DNA萃取、DNA稀釋、qPCR及定量指標 31 4.3.2.2 滅菌土壤經過1X-300X稀釋後添加未受熱與受熱原蟲進行不同濃度EMA與鹵素燈處理 31 4.3.2.2.1 製備已知濃度的未受熱與受熱原蟲懸浮液 31 4.3.2.2.2 配置1X-300X土壤稀釋液 32 4.3.2.2.3 1X-300X土壤稀釋液之總懸浮固體測量 32 4.3.2.2.4 未受熱與受熱原蟲土壤稀釋混合液配置 32 4.3.2.2.5 核酸染劑處理 33 4.3.2.2.6 DNA萃取、DNA稀釋、qPCR及定量指標 33 4.3.2.3 滅菌土壤經過10X-200X稀釋後添加未受熱與受熱原蟲進行不同濃度EMA與PhAST Blue處理 34 4.3.2.3.1 製備已知濃度受熱與未受熱棘阿米巴原蟲 34 4.3.2.3.2 配置1X-300X土壤稀釋液 34 4.3.2.3.3 未受熱與受熱原蟲土壤稀釋混合液配置 34 4.3.2.3.4 核酸染劑處理 35 4.3.2.3.5 DNA萃取、DNA稀釋、qPCR以及定量指標 36 4.3.3 生物氣膠採樣器比較 36 4.3.3.1 採樣策略 36 4.3.3.2 分析方法 37 4.4 環境驗證與暴露初探 38 4.4.1 土壤 38 4.4.1.1 採樣策略 39 4.4.1.2 分析方法 41 4.4.2 空氣 42 4.4.2.1 採樣策略 42 4.4.2.2 分析方法 43 4.4.3 水體 44 4.4.3.1 採樣策略 44 4.4.3.2 分析方法 45 4.5 土壤與空氣環境因子測量 47 4.5.1 土壤環境因子測量 47 4.5.1.1 土壤含水量測定 47 4.5.1.2 土壤pH值測定 47 4.5.1.3 土壤異營性細菌濃度測定 47 4.5.1.4 土壤植被覆蓋判定 48 4.5.1.5 土壤結構判定 49 4.5.2 空氣環境因子測量 50 4.7 資料分析 51 4.7.1 總和稀釋倍數 51 4.7.2 統計分析 51 第五章 結果 53 5.1 方法開發 53 5.1.1棘阿米巴原蟲囊體核酸染劑之選擇 53 5.1.2 EMA-qPCR定量土壤中活性棘阿米巴原蟲之方法開發 57 5.1.3 生物氣膠採樣器比較 65 5.1.4 總和稀釋倍數 67 5.2 環境驗證 69 5.3 暴露初探 71 5.3.1 土壤中總與活性棘阿米巴原蟲濃度 71 5.3.2 空氣中總與活性棘阿米巴原蟲濃度 80 5.3.3 水體中總與活性棘阿米巴原蟲濃度 83 5.3.4 土壤中環境因子測量及相關分析 86 5.5.5 空氣中環境因子測量及相關分析 103 第六章 討論 108 6.1方法開發 108 6.1.1 棘阿米巴原蟲囊體核酸染劑之選擇 108 6.1.2 EMA-qPCR定量土壤中活性棘阿米巴原蟲之方法開發 109 6.1.3 生物氣膠採樣器比較 113 6.2 暴露初探 113 6.2.1 土壤中棘阿米巴原蟲濃度與相關因子 113 6.2.2 空氣中棘阿米巴原蟲濃度與相關因子 115 6.2.3 水體中棘阿米巴原蟲濃度 117 6.3 未來研究建議 119 第七章 結論 120 第八章 參考文獻 122 附錄 132 口試委員提問與回答 148 | |
dc.language.iso | zh-TW | |
dc.title | 活性棘阿米巴原蟲定量方法開發與環境暴露初探 | zh_TW |
dc.title | Method development for quantifying viable Acanthamoeba and exposure assessment in various environments | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 曾俊傑(Chun-Chieh Tseng),李書安(Shu-An Lee) | |
dc.subject.keyword | 棘阿米巴原蟲,核酸染劑,及時定量聚合?連鎖反應,土壤,空氣,水體, | zh_TW |
dc.subject.keyword | Acanthamoeba,DNA intercalating dyes,Real-time quantitative PCR,soil,air,water, | en |
dc.relation.page | 152 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2013-01-17 | |
dc.contributor.author-college | 公共衛生學院 | zh_TW |
dc.contributor.author-dept | 環境衛生研究所 | zh_TW |
顯示於系所單位: | 環境衛生研究所 |
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