台北市污水處理廠腺病毒調查與分析之研究

THE STUDY OF ADENOVIRUS INVESTIGATION IN WASTEWATER TREATMENT PLANT IN TAIPEI CITY

張述全(Su-chung Chang)

指導教授 ： 陳建先

大同大學/工程學院/生物工程學系所/碩士(2010年)

摘要

在世界各地皆有因水體受到腺病毒的污染而造成公共衛生危害的實例發生，且腺病毒在台灣亦經常造成呼吸道感染、結膜炎與腸胃炎等感染流行。本研究室自2006年起，監測淡水河流域及北台灣海水，皆有腺病毒陽性檢出，追溯其上游區域亦有污水處理廠存在，鑒於污水處理不當有可能污染下游區域環境水體，因此本研究針對淡水河中上游區域之污水處理廠進行腺病毒調查及分析，以了解淡水河下游區腺病毒與中上游污水廠污染與否有無關聯性。
本研究首先建立一套適用於污水腺病毒回收之濃縮流洗技術，經由評估六種不同濾膜與濃縮流洗程序組合之污水腺病毒濃縮過濾技術，以帶微量負電之硝酸纖維濾膜搭配H2SO4/NaOH流洗法，於水體添加鎂離子以及pH值調整之方法為最佳，其分子生物法及細胞培養法相對回收效率皆為100%。進一步探討環境腺病毒定量檢測方法(qPCR與Plaque Assay)，其腺病毒定量之關係為1PFU/mL=2.17×104 genomic copies/mL。而使用ICC-qPCR卻可於48小時檢測出1.2PFU/mL濃度之病毒。在奈米金-PCR及奈米金-qPCR方面確實可以降低抑制物質干擾，分別提昇PCR及qPCR反應效率100倍及1.15-3.5倍左右。
接著本研究應用上述最佳病毒檢測方法於實際田野調查，結果顯示污水廠進流水存在高濃度總腺病毒(高於2.34 Log PFU/L)其來源為糞便性污染且具有感染性(高於4.25 Log PFU/L)，經Direct-PCR及Culture-PCR檢測分別可分離ADV-41及ADV-2或ADV-3得知污水中存在多種型別的腺病毒，由核酸序列分析及演化分析發現與亞太地區及台灣當地流行株有高度核酸一致性，推論台灣地區腺病毒疫情與污水腺病毒有傳播之關聯性存在。污水腺病毒不受水溫及降雨量變化的影響，但受DO、BOD、COD及SS之影響。欲了解污水腺病毒是否為糞便性污染可使用AC/TC作為指標性微生物，此外放流水指標微生物無法反應水體受到病毒污染情形，但生活污水經污水廠處理後，其總腺病毒及感染性腺病毒移除效率分別可達92±17.57%及>99%，且可降低放流水中腺病毒感染、致病及致死性風險至每年1.85×10-1、2.88×10-1及4.90×10-5。

關鍵字

污水處理廠 ； 腺病毒

並列摘要

Adenoviruses generally were spread by faecal-oral route, and the importance of viral transmission by water is probably underestimated. When people contact the contaminated food or water, it would increase the risk of public health. Since 2006, our has investigated of adenoviruses at the Damshui River and the coastal water of northern Taiwan, and the results have shown that adenoviruses were detected in those area. Large numbers of adenoviruses are excreted in human faeces, and they are known to be very abundant in raw sewage and then polluted waters. Wastewater treatment plants are the important system of monitoring pathogens transmitted to water. Thus, the objective of this study was to monitor and analyze adenoviruses in wastewater treatment plant in Taipei city in order to understand adenoviruses of correlate at downstream Damshui River and effluent water.
First, to develop a rapid and efficiency virus concentration method. Six concentration method combinations, adsorption on nitrocellulose membranes followed by an acid rinse elution consistently gave the highest recovery efficiencies. Further evaluate quantitative of adenoviruses in water samples by convention Real-Time quantitative PCR or cell culture, and new technology of ICC-qPCR or nanogold-PCR/qPCR. This study obtained 2.17×104 genomic copies/mL adenoviruses for 1 PFU/mL by qPCR and cell culture (Plaque Assay). However, using ICC-qPCR could detect 1.2 PFU/mL of adenoviruses within 48 hours. Using nanogold-PCR/qPCR could reduce inhibitors in environment sample. Au nanoparticles could increase the sensitivity of PCR detection 100-fold in nanogold-PCR and further increase 1.15-to 3.5-fold in nanogold-qPCR.
The total and infectious of adenoviruses were detected at the highest quantitative (total adenoviruses :> 2.34 Log PFU/L; infectious of adenoviruses :> 4.25 Log PFU/L) by qPCR and cell culture in swage water, furthermore sewage water was received human fecal pollution. When comparing the direct-PCR method with culture-PCR method in sewage water samples, direct-PCR just appeared that adenovirus type 41 was detected and culture-PCR revealed that there were adenovirus type 2 and 3 but none of adenovirus 41 was detected in same wastewater sample. According to the result of phylogeny tree, the result showed that the nucleotide of adenoviruses isolated from the wastewater treatment plant and had significant positive correlation with Asia-Pacific region. Therefore, the study inferred that the adenoviruses were transmitted in the Asia-Pacific region. The concentration of biochemical oxygen demand, chemical oxygen demand, settle able solid and AC/TC ratio showed significant positive correlation with the adenoviruses titers in raw sewage water. Besides, the removal efficiency of total and infectious adenoviruses also increased to 92% and >99% , respectively. However, the removal efficiency of indicator bacteria showed no correlation with adenoviruses removal efficiency in effluent water. Finally, this study proved that the risk of infection, the risk of illness and the risk of death could be reduced to 1.85×10-1, 2.88×10-1 and 4.90×10-5 per year.

並列關鍵字

wastewater treatment plant ； adenoviruses

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