- 學位論文
腺病毒定量方法評估與環境水體檢測之應用
QUANTITATIVE METHODS TO EVALUATE THE ADENOVIRUS AND THE APPLICATION OF ENVIRONMENTAL WATER TESTING
摘要
本研究以兩種新型定量方法Integrated cell culture Real-Time quantitative PCR(ICC- qPCR) )與Ethidium monoazide quantitative PCR (EMA- qPCR) )檢測環境水體中感染性腺病毒量了解其感染之風險評估。ICC- qPCR最適條件為細胞培養24 小時即可檢出腺病毒2~3 Log PFU/L 增幅，偵測腺病毒濃度範圍為3.4×105~3.4×10-1 PFU/ml。而 EMA-qPCR 最適分析條件為EMA 濃度30μg/ml，光照時間20 分鐘，最低偵測極限為0.1%活菌量，有效濃度範圍為3.37~10.7log copies/mL。將上述最適條件應用於檢測環境樣本，分別以污水處理廠與自來水體進行腺病毒檢測，污水廠各水體皆有腺病毒檢出，EMA-qPCR所檢測感染性腺病毒量隨污水廠各處理程序增加而降低，而ICCqPCR檢測結果各水體之間病毒濃度變化小。環境自來水檢測結果顯示，EMA-qPCR 感染性腺病毒檢出率僅為50%，低於其他方法檢出率，因此EMA-qPCR 不適用於檢測消毒過後水體，易造成腺病毒感染風險之低估。以EMA-qPCR 檢測污水廠放流水感染性腺病毒值為2.47 Log PFU/L，進而推估具有6.43×10-1 感染 風險、8.20×10-1致病風險及2.47×10-4致死風險，而自來水中ICC-qPCR其感染、致病與致死風險為(9.04×10-1、9.80×10-1、5.62×10-4)。2009 年污水處理廠腺病毒量變化易受物化因子(如水溫、氨氮、溶氧、餘氯、BOD 與COD)之影響，也與指標微生物大腸桿菌與總大腸桿菌具有相關性，腺病毒與感染性腺病毒移除效率分別為74.05%與99%以上。在季節變化中冬季有高濃度總腺病毒檢出量。
關鍵字
none並列摘要
In this study, there are two new quantitative methods Integrated cell culture Real-Time quantitative PCR (ICC-qPCR)) and Ethidium monoazide quantitative PCR (EMA-qPCR) to be applied to detect the infectious adenovirus in environmental water, and further to evaluate the infection risk by adenovirus. The results of optimum conditions for ICC-qPCR was appeared that the lowest detection limit for adenovirus was amplified by 2 ~ 3 log PFU / L and the detection range from 3.4 × 10-1 to 3.4 × 105 PFU / ml within 24 hours cells culture. According to the result of the optimum conditions for EMA-qPCR, the optimal concentration of EMA was 30μg/ml, and the optimal illumination time was 20 minutes. Besides, it was estimated the ratio of dead viruses to viable viruses was no more than 103, and the detection range from 3.37 to 10.7 log copies / mL. The optimum conditions for both quantative method were also applied to detect the infectious adenovirus from environmental water, including sewage water and tap water. The quantities of infectious adenovirus particle detected by EMA-qPCR method were decreased by increasing wastewater treatment procedure. There were no clear difference between three sewage treatment water for the infectious adenovirus numbers detected by ICC-qPCR method. In the light of the result of tap water, there were only 50 % detection rate by EMA-qPCR method. Therefore, the EMA-qPCR method was lower than other methods to be applied to detect infectious adenovirus in environmental water , which suggests that EMA-qPCR can not be applied to be used in the water after disinfection, and might be underestimated the adenovirus infection risk. In 2009 , the amount of sewage treatment plants adenovirus vulnerable to physical and chemical changes in factors (such as water temperature, ammonia nitrogen, dissolved oxygen, residual chlorine, BOD and COD) of the impact, but also with the indicator microorganism E. coli and total coliform Relevant bacteria. Beside, the removal efficiency of total and infectious adenovirus also increased to 74.05% and> 99%, respectively and the high concentration of the total amount of adenovirus was detected in the winter.