諾羅病毒基因非常多樣，是引起人類各個年齡層急性腸胃炎大流行及散發病例重要的病原。
本論文研究目的在於探討兒童諾羅病毒感染的疾病特徵，並分析致病諾羅病毒的基因序列及其分型。於2007至2008兩年期間從高雄地區因感染急性非細菌性腸胃炎而住院的兒科(十八歲以下)病患收集糞便檢體，若驗得輪狀病毒抗原則排除。本研究使用兩組引子對分別進行反轉錄-聚合酶連鎖反應。諾羅病毒的open reading frame 2編碼主要外殼蛋白。region C primers針對諾羅病毒的ORF2的5’端位置(編碼N端的shell domain)。region D primers針對ORF 2的3’端位置(編碼C端的高度變異區P2 subdomain)。
結果全部39份糞便檢體中以region C primers做聚合酶連鎖反應，有五位病患的檢體可驗出諾羅病毒，整體陽性率約佔12% (5/39)。其中3人年紀小於五歲，占五歲以下病童10% (3/30)。陽性檢體的5位患者皆有嘔吐症狀，其中3人有發燒。將所有PCR產物定序後上網比對，2株屬於G1.4基因型，3株屬於G2.4基因型。此五份陽性檢體再以region D primers做聚合酶連鎖反應，雖僅有一份呈陽性，但經比對後為相同基因型。
G1.4基因型為台灣文獻未曾發表過之基因型。本研究顯示，因散發的非細菌性腸胃炎而住院的五歲以下高雄地區兒童，糞便中可驗出諾羅病毒的比例為百分之十。

The noroviruses are important pathogen of epidemic and sporadic gastroenteritis in all age group and show great genetic diversity.
The aim of the present study was to describe the prevalence and genetic diversity of noroviruses among children hospitalized with acute sporadic gastroenteritis in Kaohsiung, Taiwan.
Fecal samples were collected from hospitalized pediatric patients with sporadic gastroenteritis below age of 18 years during a 2-year period (2007 to 2008). Norovirus RNA was detected by reverse transcription-polymerase chain reaction and comfirmed by sequence analysis. Two different sets of primers were used. Region C primers target shell domain at 5’ end of capsid gene and region D primers target highly variable P2 subdomain at 3’ end of capsid gene.
Noroviruses were identified in 5 of 39 (12%) rotavirus-negative specimens using region C primers. Using region D primers only one among these 5 samples could yield PCR product, which showed concordant noroviral genotype. 3 (10%, n=30) specimens from children below age of 5 years tested positive. All these 5 patients had symptoms of vomiting and 3 had fever. All PCR products were sequenced and showed 2 strains of genogroup 1 (G 1.4) and 3 strains of genogroup2 (G 2.4).
To our knowledge, this is the first report that demostrated G1.4 genotype norovirus from Taiwan. Norovirus accounted for 10% of sporadic non-bacterial gastroenteritis cases among hospitalized children below 5 years of age in Kaohsiung, Taiwan.

中文摘要...........................................................................................i
英文摘要.........................................................................................iii
英文縮寫表......................................................................................iv
壹、緒論
一、諾羅病毒流行病學..................................................................1
二、諾羅病毒的基因體結構..........................................................2
三、諾羅病毒主要外殼蛋白(VP1)的結構....................................3
四、諾羅病毒的診斷方法..............................................................4
五、諾羅病毒的分類......................................................................6
六、研究目的..................................................................................8
貳、材料與方法
一、材料..........................................................................................9
二、實驗方法..................................................................................9
1.自糞便檢體中萃取諾羅病毒的基因RNA..................................9
(1) 檢體處理....................................................................................9
(2) 核酸萃取....................................................................................9
2. 反轉錄-聚合酶連鎖反應..........................................................10
(1) 反轉錄......................................................................................10
(2) 聚合酶連鎖反應......................................................................11
3. PCR產物定序...........................................................................13
4. 序列分析...................................................................................13
參、結果
一、病患統計資料........................................................................14
二、 region C primers PCR 結果.............................................14
三、 region D primers PCR 結果.............................................14
四、檢體冷藏天數........................................................................15
五、陽性檢體病患統計資料........................................................15
肆、討論
一、急性腸胃炎兒童糞便中可驗出諾羅病毒的比例................16
二、檢體冷藏方式........................................................................16
三、不同引子PCR結果的差別...................................................17
四、諾羅病毒的序列與基因型....................................................18
伍、結論........................................................................................20
參考文獻........................................................................................21
表
表一: 實驗中使用的兩組引子對..................................................26
表二: 糞便驗出諾羅病毒的五位病患統計資料... ......................27
圖
圖一: 諾羅病毒的基因體結構及主要外殼蛋白的domain..........2
圖二: 諾羅病毒主要外殼蛋白(VP1)的domain結構....................4
圖三: 常用來設計引子偵測諾羅病毒的區域 (region A-D) ........6
圖四: 諾羅病毒的分類及命名系統................................................7
圖五: region C primers 與諾羅病毒基因體的相對位置。......12
圖六: 使用第一組(region C) primers瓊膠電泳的結果............28
附錄
附錄一: region C引子對PCR產物序列比對結果(BLAST-X)...............29
附錄二: region D引子對PCR產物序列比對結果(BLAST-X)...............39
附錄三: PCR產物定序結果.........................................................41

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