以即時聚合酶鏈鎖反應定量Acanthamoeba spp.與Hartmannella vermiformis

Quantification of Acanthamoeba spp. and Hartmannella vermiformis by real-time polymerase chain reaction

吳盈潔(Ying-Chieh Wu)

指導教授 ： 張靜文

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01926

摘要

Acanthamoeba 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。

關鍵字

棘阿米巴原蟲 ； Acanthamoeba castellanii ； Hartmannella vermiformis ； DNA萃取 ； 即時定量聚合酶鏈鎖反應

並列摘要

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.

並列關鍵字

Acanthamoeba castellanii ； Hartmannella vermiformis ； DNA extraction ； real-time quantitative polymerase chain reaction

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