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  • 學位論文

以核酸染劑結合即時定量聚合酶連鎖反應 定量活性Acanthamoeba

Quantification of viable Acanthamoeba by real-time quantitative PCR coupled with DNA binding dyes

指導教授 : 張靜文

摘要


棘阿米巴(Acanthamoeba)廣泛存在於各種環境中。暴露於活性棘阿米巴可能有感染棘阿米巴角膜炎(Acanthamoeba keratitis, AK)、棘阿米巴皮膚炎(cutaneous acanthamebiasis)與棘阿米巴腦膜炎(Acanthamoeba granulomatous encephalitis)之風險。為能掌控棘阿米巴對人類健康造成的風險,定量環境中活性棘阿米巴扮演著重要的角色。據此,本研究嘗試開發以即時定量聚合酶連鎖反應(real-time quantitative polymerase chain reaction, qPCR)搭配核酸染劑(ethidium monoazide, EMA與propidium monoazide, PMA),用於檢測並定量活性棘阿米巴之18S rRNA基因。   透過螢光顯微鏡搭配23 μg/mL EMA之染色處理,本研究證實EMA可顯著進入非活性棘阿米巴滋養體(trophozoites)與囊體(cysts)體內,具備區分活性與非活性棘阿米巴之能力。在此基礎下,本研究將活性與非活性棘阿米巴滋養體分別以不同濃度(2.3、23或76.7 μg/mL)之EMA或PMA反應5分鐘,搭配500瓦特鹵素燈照光5、10或20分鐘,再經由qPCR定量DNA分析以評估最佳核酸染劑處理條件。結果顯示EMA與PMA對於定量活性與非活性棘阿米巴之能力並無統計上明顯差異(P = 0.66與P = 0.17)。且相較於無核酸染劑處理之活性棘阿米巴,兩種核酸染劑處理後所得之之DNA量亦無顯著差異(P = 0.78與P = 0.13),然兩種染劑可顯著抑制非活性棘阿米巴之DNA被qPCR偵測與定量(P = 0.02與P = 0.0001)。其中EMA之各濃度與各照光時間在DNA量並無統計差別。據此,本研究以2. 3 μg/mL EMA反應5分鐘,搭配500瓦特鹵素燈照光20分鐘作為棘阿米巴之最佳核酸染劑處理之條件。而此方法於線性關性良好(R2 = 0.98)情況下,定量活性棘阿米巴之偵測上限為1.5 x 105 cells,下限則為1.5 x 102 cells。本研究也發現,使用此最佳核酸染劑處理條件可顯著區分活性與面對不同受熱溫度(75、80、85、90與95°C)下不同囊化程度之棘阿米巴,包括未完全囊化之棘阿米巴(囊體百分比為53.6 ± 3.49%)(P < 0.0001)與棘阿米巴囊體(囊體百分比為91.7 ± 1.79%)(P < 0.0001)。且受熱溫度與qPCR所得之DNA量呈現劑量效應之關係(r = -0.95, P < 0.0001;r = -0.93, P < 0.0001)。比較滋養體與不同囊化程度之A. castellanii則顯示,A. castellanii 囊體對熱之耐受程度最高, 未完全囊化之 A. castellanii次之,耐熱程度最低者為A. castellanii 滋養體。

並列摘要


Acanthamoeba has been detected from various natural and artificial environments. Exposure to viable Acanthamoeba may cause severe Acanthamoeba keratitis (AK) which could result in blindness, cutaneous acanthamebiasis and lethal Acanthamoeba granulomatous encephalitis (AGE) in humans. Accurate quantification of viable Acanthamoeba plays an important role to assess the risk that Acanthamoeba poses to human health. Hence, we have adopted ethidium monoazide (EMA) and propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) to rapidly detect and quantify viable Acanthamoeba exclusively.   With alive and heat-inactivated Acanthamoeba trophozoites and cysts stained with 23μg/mL EMA and analyzed by epifluorescence microscopy, we found that heat-inactivated Acanthamoeba were significantly permeable to EMA and were stained bright red. The result showed EMA had the ability of discriminating between alive and heat-inactivated Acanthamoeba. Base on the finding, alive and heat-inactivated cells were treated with EMA or PMA in nine combinations at 2.3, 23 or 76.7 μg/mL with a 500-W light exposure for 5, 10 or 20 min. Alive and heat-inactivated cells were also prepared without EMA or PMA treatment as controls. After DNA extraction and qPCR analysis, DNA quantity in controls and samples was determined. The results indicated that there is no statistically significant difference between the use of EMA and PMA in quantifying alive and heat-inactivated Acanthamoeba (P = 0.66, P = 0.17). Further analysis of alive cells, our results showed no statistically significant difference (P = 0.78, P = 0.13) in DNA quantity between EMA/PMA-treated and -untreated samples, whereas a significant decrease of DNA quantity (P = 0.02, P = 0.0001) was observed for the heat-inactivated cells pretreated with EMA or PMA as compared to that of deal cells without EMA/PMA treatment. The DNA quantity in heat-inactivated cells of nine EMA combinations was statistically indifferent. According to the DNA quantity and statistical results, the optimal protocol was determined as cells incubated 2.3 μg/mL EMA for 5 min with a 500-W light exposure for 20 min. Under a good linearity (R2 = 0.98) for the method, the upper and lower detection limits for quantification viable Acanthamoeba were 1.5 x 105 cells and 1.5 x 102 cells respectively. Our results also demonstrated that the optimal protocol can significantly differentiate alive and heat-inactivated (75, 80, 85, 90 and 95°C, 20 min) Transient Acanthamoeba (cyst percentage: 53.6±3.49%) (P < 0.0001) and Acanthamoeba cysts (cyst percentage: 91.7±1.79%) (P < 0.0001). Moreover, the DNA quantity was decreased with the increasing level of heat stress for EMA-treated Transient Acanthamoeba and Acanthamoeba cysts in a dose-response matter (r = -0.95 and -0.93, respectively, both P < 0.0001). Our results demonstrated that Acanthamoeba trophozoites were the most sensitive to heat stress, followed by Transient Acanthamoeba, and the last were Acanthamoeba cysts.

參考文獻


1.Aksozek A, McClellan K, Howard K, Niederkorn JY & Alizadeh H (2002) Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J Parasitol 88: 621-623.
2.Allen PG & Dawidowicz EA (1990) Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast. J Cell Physiol 145: 508-513.
3.Bae S & Wuertz S (2009) Discrimination of viable and dead fecal Bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl Environ Microbiol 75: 2940-2944.
4.Banning N, Toze S & Mee BJ (2002) Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein. J Appl Microbiol 93: 69-76.
5.Barer MR, Smith RJ, Cooney RP & Kimmitt PT (2000) Relationships between culturability, activity and virulence in pathogenic bacteria. J Infect Chemother 6: 108-111.

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