張靜文Chang, Ching-Wen臺灣大學:環境衛生研究所吳盈潔Wu, Ying-ChiehYing-ChiehWu2010-05-072018-06-302010-05-072018-06-302008U0001-2407200819130300http://ntur.lib.ntu.edu.tw//handle/246246/181413Acanthamoeba spp.及Hartmannella vermiformis為自營性阿米巴原蟲，常存在於水體環境中，不僅本身具有致病性，且為致病菌繁殖增生的宿主。本研究針對此兩種原蟲，建立一套應用即時定量聚合酶鏈鎖反應 (real-time quantitative polymerase chain reaction, real-time qPCR) 之定量方法。 為了選出一種可同時應用於Acanthamoeba spp.及H. vermiformis之DNA萃取試劑，本研究比較兩種常用者: Wizard® SV genomic DNA purification system (Promega) 及FastDNA spin kit for soil (MP) 搭配real-time qPCR進行分析結果，發現其各自最佳之萃取流程為Promega使用離心法，而MP需增加震盪時間及沖提體積。另採集冷卻水塔水樣、水塔內壁生物膜 (substrate biofilm，SB) 及水塔內空氣與水交界處的生物膜 (floating biofilm，FB)，評估此兩種DNA萃取試劑在環境樣本之適用性。結果顯示以MP萃取水樣、SB與FB之DNA並分別稀釋100倍、100倍、10倍後，real-time qPCR偵測出的Acanthamoeba DNA含量均分別較Promega萃取者高0.7、0.5、0.8 log fg；MP萃取後所能偵測之H. vermiformis DNA量亦分別較Promega多1.3、1.1、1.4 log fg。此外，以MP萃取之環境樣本，Acanthamoeba spp.及H. vermiformis的陽性數於各式環境樣本亦較高，顯示MP較Promega適於萃取環境樣本。進一步以MP萃取Acanthamoeba castellanii與H. vermiformis營養體及囊體之DNA，評估水樣及FB樣本採樣後之前處理流程對DNA定量的影響。以無前處理者為100%，結果顯示於水樣及FB樣本的DNA相對回收率：A. castellanii營養體為88.10±8.48%及65.93±10.96%，而囊體則為110.09±22.95%及94.69±7.25%；H. vermiformis營養體為108.14±13.08%及75.07±31.24%，囊體為137.15±72.25%及78.31±17.04%，顯示水樣之回收率均較FB高。此外，本研究也建立以A. castellanii或H. vermiformis原蟲數對應DNA量的檢量線，其偵測下限最低可達3原蟲數。 綜合上述結果，本研究開發以MP結合適當稀釋倍數與real-time qPCR應用於定量環境中的Acanthamoeba spp.及H. vermiformis的方法，藉此未來除可快速了解此類原蟲在環境水體的分布，亦可進一步評估其與他種寄生致病菌間之消長關係。Free-living amoebae (FLA) of Acanthamoeba spp. and Hartmannella vermiformis are widely distributed in various aquatic habitats. They are the hosts of many pathogenic bacteria and have been found to cause opportunistic infection. In this study, we developed a real-time quantitative polymerase chain reaction (real-time qPCR) to target these two types of amoebae. For selecting a better DNA extraction kit, we compared two commercial DNA extraction kits, Wizard® SV genomic DNA purification system (Promega) and FastDNA spin kit for soil (MP).We optimized each protocol of these two kits at first, and the data showed it was better when using microcentrifuge method for Promega and increasing both vortex time and elution volume for MP. Both kits were further coupled with real-time qPCR assay to determine the DNA quantity of Acanthamoeba spp. and H. vermiformis from the samples prepared in lab and collected from water, substrate biofilm (SB) or floating biofilm (FB) of cooling towers. After 100-fold, 100-fold, and 10-fold dilution of DNA extracted from water, SB, and FB samples, respectively, and determination by real-time qPCR, quantity of Acanthamoeba DNA extracted by MP was 0.7, 0.5, and 0.8 log fg, respectively, more than that by Promega. Similarity, H. vermiformis DNA extracted by MP was also 1.3, 1.1, and 1.4 log fg greater than that by Promega, respectively. Moreover, the number of amoebae-positive samples was generally greater in MP-extracted samples than that in Promega-extracted ones regardless of sample type or amoebic genera, indicating MP is more applicable for environmental samples than Promega. We further adopted MP to extract DNA from trophozoites and cysts of A. castellanii and H. vermiformis to evaluate the effect of pretreatment in water and FB samples on DNA quantity. With the DNA extracted from the sample without pretreatment as reference, the relative DNA recovery rates of water and FB samples were 88.10±8.48% and 65.93±10.96% for A. castellanii trophozoite, respectively, and 110.09±22.95% and 94.69±7.25% for A. castellanii cyst, respectively. As for H. vermiformis, the respective recovery rates of water and FB samples were 108.14±13.08% and 75.07±31.24% for trophozoite and 137.15±72.25% and 78.31±17.04% for cyst. These results indicate the DNA recovery rate of water samples was greater than that of FB samples for both of A. castellanii and H. vermiformis. Finally, a calibration curve between amoebic number and DNA quantity was determined, from which the detection limit was found as low as 3 amoebae. In conclusion, the present study showed MP kit coupled with appropriate DNA dilution and real-time qPCR assay is an optimal method to quantify Acanthamoeba spp. and H. vermiformis from artificial water reservoirs. This methodology provides the potential to accurately characterize the distribution of these two types of amoebae, and may be further used to evaluate the relationship between the amoebae and their parasitic pathogens.第一章　前言 1二章　文獻回顧 2.1.阿米巴原蟲 (amoeba) 2.1.1. Acanthamoeba spp. (棘阿米巴原蟲) 2.1.2. Hartmannella vermiformis 3.2. 冷卻水塔 3.3. 原蟲偵測方法 4.4. 研究的重要性 7三章　研究目的 8四章 研究架構 9.1. DNA萃取流程最佳化 10.2. 選取最適DNA萃取試劑 11.3. 評估DNA萃取試劑應用於冷卻水塔樣本之DNA相對回收率及偵測下限 13五章 材料與方法 14.1. 實驗物種 14.1.1. A. castellanii 14.1.2. H. vermiformis 14.2. 培養基及緩衝溶液製備 15.2.1. ATCC medium 712 15.2.2. ATCC medium 1034 16.2.3. 囊化培養基 16.2.4. Page’s Amoeba Saline (PAS) 17.2.5. 磷酸緩衝液 (phosphate buffered saline, PBS) 18.2.6. TE buffer 18.3. DNA萃取最佳化 19.3.1. Promega萃取效率評估 19.3.2. MP萃取效率評估 22.3.3. Promega與MP比較 24.3.4. DNA相對回收率及偵測下限 32.4. Real-time qPCR 35.4.1. Acanthamoeba spp. 35.4.2. H. vermiformis 37.4.3. 標準品製備及PCR複製效率 39.4. 統計分析 40六章　結果 41.1. 原蟲型態 41.1.1. A. castellanii 41.1.2. H. vermiformis 43.2. DNA萃取試劑最佳化 45.2.1. Promega萃取效率評估 45.2.2. MP萃取效率評估 46.3. A. castellanii 定量 48.3.1. DNA純度 48.3.2. DNA-based calibration curve 49.3.3. Cell-based calibration curve 50.3.4. 環境樣本適用性評估 53.3.5. DNA相對回收率 57.3.6. 水樣與FB樣本偵測下限 57.3.7. Real-time qPCR QA/QC 63.4. H. vermiformis定量 66.4.1. DNA純度 66.4.2. DNA-based calibration curve 68.4.3. Cell-based calibration curve 69.4.4. 環境樣本適用性評估 73.4.5. DNA相對回收率 76.4.6. 水樣及FB樣本偵測下限 77.4.7. Real-time qPCR QA/QC 83.5. 統計檢定 92七章　討論 96.1. Promega與MP比較 96.1.1. DNA純度 96.1.2. DNA-based calibration curve 96.1.3. Cell-based calibration curve 97.1.4. 營養體與囊體之PCR比較 98.1.5. 冷卻水塔環境應用性 99.1.6. Promega與MP之比較 99.2. Acanthamoeba spp. 100.2.1. 前處理對DNA回收率的影響 100.2.2. 偵測下限 101.2.3. 冷卻水塔Acanthamoeba spp.濃度 101.2.4. PCR引子對及探針特異性 101.3. H. vermiformis 102.3.1. 偵測下限 102.3.2. 環境樣本萃取後之DNA稀釋 103.3.3. 冷卻水塔環境樣本應用性 103.3.4. PCR引子對特異性 104.4. SB檢量線 104.5. DNA萃取與PCR分析條件 105八章 結論與建議 108考文獻 110錄 117application/pdf4347491 bytesapplication/pdfen-US棘阿米巴原蟲Acanthamoeba castellaniiHartmannella vermiformisDNA萃取即時定量聚合酶鏈鎖反應DNA extractionreal-time quantitative polymerase chain reactionthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181413/1/ntu-97-R95844008-1.pdf