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研究生：	劉家全 Liu, Chia-Chuan
論文名稱：	台灣南部腸病毒疫情流行區水中腸病毒之分子生物學及其可能傳播路徑研究 The Study of Molecular Biology and Possible Spreading Pathway for the Enteroviruses in Water of the Southern Taiwan Enteroviruses Epidemic Areas
指導教授：	簡錦樹 Jean, Jiin-Shuh
學位類別：	碩士 Master
系所名稱：	理學院 - 地球科學系 Department of Earth Sciences
論文出版年：	2002
畢業學年度：	90
語文別：	中文
論文頁數：	93
中文關鍵詞：	污水傳播路徑、地下水、腸病毒71型、柯沙奇病毒、分子生物、腸病毒
外文關鍵詞：	EV71, enteroviruses, molecular biology techniques, coxackieviruses, spreading pathway, groundwater, sewage water
相關次數：	點閱：82 下載：2
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摘要
本研究目的主要從腸病毒疫情流行區的地下水及污水中分離出腸病毒, 並以分子生物技術鑑定之, 以了解腸病毒在自然環境間的可能傳播路徑。本研究的細胞培養病毒接種結果顯示有細胞病變(cpe)反應，其代表了2001年6月4日在高雄縣阿蓮鄉所採集的地下水及污水樣有病毒存在的反應。另外，濃縮之水樣在經過了RT-PCR、膠電泳實驗後，可以發現在1998年9月20日在屏東縣鹽埔鄉所採集的地下水樣濃縮液中，有腸病毒的存在。配合屏東縣鹽埔鄉的水文地質剖面圖、時降雨強度、本研究所採集地下水樣的時間及患者發病日期(9/19/1998)等，得到的結果說明了此區腸病毒的傳播，大約是降雨強度在30mm/hour以上的大雨後9-11日可在抽水井處得到含有腸病毒之地下水樣，而腸病毒在地下水中傳播的速度隨著降雨強度、抽水率及抽水影響範圍、水井與污染源距離、含水層之水力傳導度及孔隙率、水力梯度、土壤吸附程度、水力延散、地下水深度等之不同而變。本研究乃針對了近幾年常見的腸病毒種類及對人類危害性較大的種類選出了柯沙奇病毒(coxackieviruses) A16型﹑B1型﹑B3型﹑B5型, 以及腸病毒71型(EV71)等五株的腸病毒作為研究。

ABSTRACT
The purpose of this study was to isolate the enteroviruses from groundwater and sewage water in the enteroviruses epidemic areas. The enteroviruses were identified by molecular biology technologies in order to understand the spreading pathway of enteroviruses in the environment. The results of cell culture and virus inoculation showed the cytopathic effect(cpe) of enteroviruses for the groundwater samples collected on June 4, 2000 from Alien County, Kaohsiung. In addition, the enteroviruses in the concentrated water, collected on September 20,1998 from the groundwater in a certain area of Yen-Pu County, Pingtung, were detected by the RT-PCR and gel electrophoresis procedures. In association with the hydrogeological profile, hourly rainfall intensity, and the groundwater sampling time, and the onset date of a patient(9/19/1998), it indicated that the enteroviruses were eluted into groundwater and started to spread after a heavy rainfall with a rainfall intensity greater than 30mm/hour, After the elapsed time of 9 to 11 days transport, the enteroviruses-
contained groundwater sample can be obtained from a pumped well. The spreading speed of enterovirus in groundwater varies with the rainfall intensity, pumping rate and its radius of influence, the distance between a well and a contaminant source, the aquifer hydraulic conductivity, porosity, and hydraulic gradient, the degree of soil adsorption, hydrodynamic dispersion, groundwater depth,etc.The five strains of enteroviruses(i.e.,coxsackie-
viruses A16, B1, B3, B5 and EV71) were selected for this study based upon the commonly found enteroviruses in recent years, which were more pernicious to humans.

目 錄                   
摘  要-------------------------------------------------------I
Abstract----------------------------------------------------II
致  謝------------------------------------------------------IV
目  錄-------------------------------------------------------V
表目錄----------------------------------------------------VIII
圖目錄------------------------------------------------------IX
第一章 緒論
        1-1 研究動機-----------------------------------------1
1-2 研究目的-----------------------------------------2
1-3 腸病毒疫情及腸病毒特性之概述---------------------2
1-4 文獻回顧-----------------------------------------5
1-5 研究範圍----------------------------------------12
第二章  研究方法
        2-1 研究流程----------------------------------------13
2-2 水樣採集過濾及病毒之萃取、濃縮------------------14
2-2-1 滅菌方式----------------------------------14
2-2-2 實驗器材及裝置----------------------------15
2-2-3 實驗藥品及溶液配製------------------------16
2-2-4 水樣採集過濾------------------------------17
2-2-4-1 採集地點之水文地質概況--------------17
2-2-4-2 水樣過濾----------------------------21
2-2-5 病毒萃取(Virus Elution)-------------------22
2-2-6 病毒濃縮(Virus Concentration)-------------23
2-3 穿透式電子顯微鏡(TEM) 之銅網樣本製備------------24
2-4  腸病毒的細胞培養與接種-------------------------25
            2-4-1 使用之細胞種類----------------------------25
2-4-2 實驗器材、藥品及溶液配製------------------26
2-4-3 細胞培養----------------------------------27
2-4-4 病毒接種----------------------------------28
2-4-5 病毒收集----------------------------------29
2-5 分子生物技術鑑定--------------------------------30
2-5-1 RNA萃取-----------------------------------30
2-5-2 逆轉錄—聚合酶連鎖反應(RT-PCR)------------31
2-5-3 膠電泳實驗--------------------------------34
2-5-4 實驗器材、藥品及溶液配置------------------35
2-5-5 腸病毒引子(primer)之設計------------------37
2-5-6 腸病毒引子(primer)之驗證------------------39
第三章 結果
3-1 腸病毒之電子影像--------------------------------41
3-2 腸病毒培養之細胞病變反應------------------------41
3-3 腸病毒在膠電泳槽之分布--------------------------42
3-4 腸病毒疫情與環境間之關係------------------------43
第四章  討論
4-1 腸病毒之可能來源及傳播路徑----------------------50
4-2 影響腸病毒傳播之可能的環境因子------------------51
4-3 腸病毒疫情與環境間之關係------------------------61
4-4 腸病毒水樣採集﹑過濾及濃縮之妥適設計------------62
第五章	  結論------------------------------------------------66
參考文獻----------------------------------------------------68
附錄一 Human coxsackievirus B1核苷酸序列------------------------73
附錄二 Human coxsackievirus B3核苷酸序列-----------------------77
附錄三 Human coxsackievirus B5核苷酸序列-----------------------81
附錄四 Human coxsackievirus A16核苷酸序列----------------------85
附錄五 Human enterovirus 71 核苷酸序列-----------------------89
自述 -----------------------------------------------------------------------------------------93
        

                                    

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校內：2004-07-22公開
校外：2004-07-22公開

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