臺灣博碩士論文加值系統

摘 要
輪狀病毒，腸腺病毒，杯狀病毒和腸病毒是引發急性腸胃炎相關的病毒。 這些腸胃炎病毒在台灣地區所扮演的角色仍是未知的。而且，在台灣7月到9月是盛行期也是輪狀病毒與腸病毒對於幼小孩童會造成混合感染的時期。 因此，本研究的目的是以分子生物學的方法(RT-PCR和ELISA)調查輪狀病毒與腸病毒在台灣的交叉流行期分布。
在台灣的腸病毒感染盛行期間，從全台各地的臨床實驗室總共收集了88個糞便檢體。每個檢體都先經過輪狀病毒的ELISA kit測試，然後再以RT-PCR來確認輪狀病毒與腸病毒的型別。
在88個具有腹瀉症狀的孩童糞便檢體中，有32個檢體呈現腸病毒陽性(佔36.4%) ，10個檢體呈現輪狀病毒陽性(佔11.4%)，而其中有3個檢體是具有兩種腸內病毒的混合感染(佔3.4%)。根據台灣的地理環境來觀察，輪狀病毒和腸病毒在北台灣較為流行，分別檢測出7個和21個陽性檢體；之後是中台灣，分別檢測出3個和10個陽性檢體；然後是南台灣，只有檢測出ㄧ個腸病毒的陽性檢體，輪狀病毒則無陽性反應。所有經輪狀病毒ELISA kit偵測所得到的陽性檢體之結果與輪狀病毒的RT-PCR結果相互吻合，證實RT-PCR之可行性。而且，在兩種方法之間沒有顯著的統計上的差異。 因此，證實RT-PCR在臨床糞便檢體之輪狀病毒檢測上，具有相同的效率。
在腸病毒的檢測中，以腸病毒71型的檢出率最高(佔47%)，其次為Coxsackievirus A(簡稱CA)的CA16 (佔38%)；而CA6與Echovirus(簡稱ECV) 的ECV 11分別檢測出兩個陽性檢體，而在本實驗中也檢測出1個檢體隸屬CA5型。在輪狀病毒的檢測中，將其陽性結果之基因序列與輪狀病毒的VP 7基因序列進行比對，其中有7個檢體的比對結果為輪狀病毒G3血清型，其相似度分別達98-99%；其餘檢體分別比對出2個G1與1個G2血清型別，其相似度分別達91-99%不等。以親緣演化的分析可發現其輪狀病毒的發生感染都在東亞地區(中國，日本和台灣)作循環。

ABSTART
The etiologic agents related to acute gastroenteritis are rotavirus, enteric adenovirus, calicivirus, and enterovirus. It is still unknown what the role of these gastroenteritis viruses in Taiwan is. Besides, there were one peak season (July-September) regarded as mix infection for enterovirus and rotavirus for younger children population in Taiwan. Therefore, the object of this study is to investigate the prevalence of both enteric virus in Taiwan by molecular method (RT-PCR and ELISA).
Total 88 fecal samples were collected from clinical laboratories distributed in different parts of Taiwan during enterovirus outbreak period in Taiwan. Each sample was first detected by rotavirus specific ELISA kit, and then the molecular typing of enterovirus and rotavirus was confirmed by reverse transcription-PCR.
Among 88 fecal specimens collected from diarrheic children, 32 (36.4%) were positive for enterovirus, 10(11.4%) were for rotavirus and 3 (3.4%) patients were presented mix infection for both two enteric virus. According to geographic distribution, enterovirus and rotavirus were more prevalent in Northern Taiwan (21,7), followed by Central Taiwan (10,3) and then Southern Taiwan (1,0). All positive samples for rotavirus ELISA assay were also showed as positive for RT-PCR method. Besides, there was no significant statistical difference between two method. Therefore, RT-PCR method could perform same efficiency for detection of rotavirus in clinical stool samples.
The most prevalent genotype were Enterovirus 71 (47 %), followed by coxsackievirus A 16(38%), and then CA6 and Ecovirus 11 were detected in two sample. We also find one sample was identified as CA5. Comparison of the nucleotide sequence with the VP7 gene of these rotavirus strains revealed that seven strains resembled closely with G3 serotype, the homology being 98 and 99 %, respectively. The following genotype was G1 for two samples and G2 for only one sample, shared 91-99 %. Phylogenetic analysis suggested that rotavirus were circulated in East Asia region (China, Japan and Taiwan).

目錄
致謝 i
中文摘要 ii
英文摘要…….…………….........………………………….……………iv
目錄….…………………......………………………………...………….vi
圖目錄………………….......……………………………..……………. ix
表目錄 ………………………………………………………………….x
第一章 緒論…………………………………………………………… 1
1.1動機……………………………………………………………… 1
1.2文獻回顧………………………………………………………… 2
1.2.1輪狀病毒……………………………………………………2
1.2.1.1輪狀病毒簡介……………………………………… 2
1.2.1.2輪狀病毒的流行病學……………………………… 4
1.2.1.3輪狀病毒的感染途徑與臨床表徵………………… 7
1.2.1.4輪狀病毒的檢測方法……………………………… 8
1.2.2腸病毒…………………………………………………… 14
1.2.2.1腸病毒簡介 ……………………………………14
1.2.2.2 腸病毒的流行病學……………………………… 15
1.2.2.3 腸病毒的感染途徑與臨床表徵………………… 16
1.2.2.4 腸病毒的檢測方法……………………………… 17
1.3 本文目的………………………………………………………21
第二章 材料與方法………………………………………………… 22
2.1 檢體來源………………………………………………………22
2.2 正、負控制組之選定…………………………………………22
2.2.1 輪狀病毒………………………………………………… 22
2.2.2 腸病毒…………………………………………………… 22
2.3 實驗試劑………………………………………………………24
2.3.1 Rotavirus ELISA Kit…………………………………….24
2.3.2 病毒核酸萃取試劑……………………………………… 24
2.3.3聚合酵素鏈鎖反應試劑 …………………………………24
2.4 實驗方法………………………………………………………24
2.5 檢體前處理……………………………………………………25
2.6 病毒核酸萃取…………………………………………………28
2.7 聚合酵素鏈所反應(PCR)……………………………………..29
2.7.1 本實驗所使用之引子…………………………………… 29
2.7.2 PCR反應溶液組成..........................................................30
2.7.2.1 RT-PCR…………………………………………… 30
2.7.2.1.1 輪狀病毒...........................................................30
2.7.2.1.2 腸病毒 ……………………………………… 31
2.7.2.2 Seminest PCR ………………………………… 32
2.7.2.2.1輪狀病毒………………………………………32
2.7.2.2.2 腸病毒……………………………………… 32
2.8 核酸序列分析……………………………………………… 39
2.8.1 核酸定序 …………………………………………………39
2.8.2 親緣演化分析 ……………………………………………39
第三章 結果與討論 ………………………………………………40
3.1 比較RT-PCR與酵素免疫分析法對輪狀病毒檢出率之評
估 …………………………………………………………40
3.2 台灣地區輪狀病毒與腸病毒之檢出情形 ……………… 50
3.3 7-9月各月份輪狀病毒與腸病毒之檢出情形 …………… 53
3.4 輪狀病毒與腸病毒之型別分析 ………………………… 55
3.5 台灣各地區輪狀病毒與腸病毒之型別相對盛行情形…… 58
3.6 探討7-9月份輪狀病毒與腸病毒之型別相對減出情形 …60
3.7 輪狀病毒與腸病毒之序列分析…………………………… 62
3.7.1 輪狀病毒 ………………………………………………62
3.7.2 腸病毒 …………………………………………………64
3.8 輪狀病毒與腸病毒交叉感染之發生 …………………… 66
第四章 結論 …………………………………………………… 68
參考文獻 ………………………………………………………… 70
圖目錄
圖1.1 輪狀病毒以電子顯微鏡觀察之電顯圖……...…………..……. 12
圖1.2 輪狀病毒剖析圖 ……………………………………………… 13
圖1.3 腸病毒署之分類 ……………………………………………… 19
圖1.4 腸病毒以電子顯微鏡觀察之電顯圖……………...……………20
圖2.1 BMD-Rotavirus rapid immuno-chromatography test之操作流
程與結果判讀 ………………………………………………… 26
圖2.2 本研究之實驗分析流程圖…………………………………….. 27
圖3.1 Lee method之Seminest PCR其各產物大小…………………. 43
圖3.2 Lee method的Seminest PCR電泳圖結果( I ) ….……………..44
圖3.3 Lee method的Seminest PCR電泳圖結果( II ) ………………..44
圖3.4 Daniel method的PCR電泳圖結果( I ) ………………………..45
圖3.5 Daniel method的RT-PCR電泳圖結果(II) …………………46
圖3.6 台灣地區輪狀病毒與腸病毒各型別之感染分布情形 ……… 59
圖3.7 輪狀病毒與腸病毒各型別在7-9月份之感染分布情形………61
圖3.8 2005年7月至9月本研究所分離之輪狀病毒VP7基因核酸
序列之親緣樹狀圖 …………………………………………… 63
圖3.9 2005年7月至9月本研究所分離之腸病毒5’UTR基因核酸
序列之親緣樹狀圖………………………………………………65
表目錄
表1.1 Lee method與Daniel method之方法比較……………………11
表2.1 各檢體送檢來源 ………………………………………………23
表2.2 本研究所使用之引子……...………..………………………… 29
表2.3 輪狀病毒One Step RT-PCR試劑配製( I )…………………….33
表2.4 輪狀病毒One Step RT-PCR試劑配製( II ) …………………...34
表2.5 腸病毒One Step RT-PCR試劑配製……….…………………..35
表2.6 輪狀病毒Seminest PCR試劑配製( I ) ………………………..36
表2.7 輪狀病毒Seminest PCR試劑配製( II ) ……………………….36
表2.8 腸病毒Seminest PCR試劑配製……...………………………..37
表2.9 本研究所使用之PCR條件…………………………………… 38
表3.1 檢測輪狀病毒之方法比較 ……………………………………47
表3.2 輪狀病毒陽性株之結果總覽 …………………………………48
表3.3 Lee method與Daniel method之結果比較……………………49
表3.4 台灣地區輪狀病毒與腸病毒的感染分布情形………………. 52
表3.5 輪狀病毒與腸病毒在7-9月份的感染分布情形 …………... 54
表3.6 臨床分離株與輪狀病毒親近株比對之結果………………… 56
表3.7 臨床分離株與腸病毒親近株比對之結果…………………… 57
表3.8 台灣地區輪狀病毒與腸病毒各型別感染分布情形………… 61

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