以分子生物學法進行胃腸炎症狀相關病毒之糞便檢體研究

THE STUDY OF MOLECULAR BIOLOGY METHOD FOR THE GASTROENTERITIS SYNDROME IN STOOL

戴鈺芬(Yu-Fen Tai)

指導教授 ： 陳建先

大同大學/工程學院/生物工程學系所/碩士(2008年)

摘要

中文摘要
腺病毒、輪狀病毒、諾羅病毒、星狀病毒與腸病毒等皆是引發急性胃腸炎的相關病毒，感染的族群主要為孩童，主要經由糞口傳播。因此，為了解台灣地區造成急性胃腸炎之各種病毒因子在不同地區、季節及年齡之流行狀況，收集各地區之急性胃腸炎病毒之糞便檢體來進行檢測，本研究針對2005年7月至2007年7月臨床糞便檢體的胃腸炎病毒進行調查。檢體樣本之分析方法採用ELISA、RT-PCR、半巢式-PCR與細胞培養法，統計數據採用One-way ANOVA方法來進行台灣各地區、季節、年齡之檢測結果，並探討所收集臨床檢體之各種胃腸炎病毒之分佈情形。
本研究所偵測的胃腸炎病毒為腺病毒、輪狀病毒、諾羅病毒，總共收集了北（219）、中（24）、南（27）三個地區共270件糞便檢體樣本。檢出病毒陽性之檢體中輪狀病毒有50個（18.5%），諾羅病毒有48個（佔17.7%），而腺病毒僅有9個（佔3.3%）。此外有16個糞便檢體呈現不同病毒交叉感染（佔5.9%）(輪狀病毒、腺病毒與諾羅病毒)，交叉病毒以輪狀病毒與諾羅病毒GI型佔31.2%（5/16）的檢出率較高，其次為輪狀病毒與諾羅病毒GII型佔25%（4/16）。
就胃腸炎病毒患者年齡分析之結果發現，患者均屬幼兒(0~10歲) 居多如輪狀病毒有60％（30/50），腺病毒為88.8%（8/9），諾羅病毒GI 與GII亦各分佈81.8％（18/22）及67.7%（21/31）的幼童，經由One-Way ANOVA test（Analysis Of Variance）分析證實感染急性胃腸炎病毒與年齡有統計相關性。然而病毒與地區分布卻無顯著統計相關性。
而就病毒檢測方法比較，本研究同時比較RT-PCR與半巢式-PCR之偵測靈敏性，此一結果顯示半巢式-PCR較能真實的反映各種胃腸炎病毒的檢出情形。就台灣地區腹瀉患者與病毒感染之相關性分析，亦經由本研究經由單方變異統計檢定P <0.05，所以驗證得知腹瀉患者與感染胃腸炎病毒是有相關性。
再以台灣地區胃腸炎之流行季節分析，發現春季的胃腸炎病毒以諾羅病毒的GI檢出率最高31.8％，夏天為腺病毒77.7％，秋天則是輪狀病毒檢出率最高30％，而交叉病毒感染的季節則是以秋季檢出率最高為43.7％，細分流行月份亦證實7~9月為高胃腸炎病毒陽性檢出數(>15人/月)。
綜合本研究於2005年7月至2007年7月年間收集107胃腸炎病毒引發之糞便檢體，經由核酸定序與NCBI GeneBank 進行BLAST比對後，發現本研究的胃腸炎病毒中的輪狀病毒分屬G1、G3及G9等型別，腺病毒則為ADV-3、ADV-41及ADV-2等型別，而諾羅病毒中分屬GI/8及GII/4等。又經由分子演化分析後顯示與東亞地區2005-2006的流行病毒株來源上具有一致性，此顯示了台灣地區胃腸炎病毒疫情確實與週遭東亞區域環境的傳播有一定程度的關連。
關鍵字:輪狀病毒, 腺病毒, 諾羅病毒

關鍵字

輪狀病毒 ； 腺病毒 ； 諾羅病毒

並列摘要

ABSTART
Adenovirus, rotavirus, norovirus, astrovirus, and enterovirus are the major viruses in causing acute gastroenteritis among human beings. Especially for children is higher risk to infect than adults. Therefore, to evaluate the major parameter to effect on acute gastroenteritis, we collect stool samples from northern to southern Taiwan of 270 fecal specimens of diarrhea patients, since July 2005 until July 2007. The study collection specimen’s direction method used ELISA, RT-PCR, Seminest-PCR.
Among 270 fecal specimens collected from diarrhea patients, 50 (18.5%) were positive for rotavirus, 48(17.7%) were positive for norovirus and 9 (3.3%) patients were detected as positive for adenovirus. Besides, dual infections were found in 5.9 % (16/270) of the positive samples for two of three enteric viruses. There were five diarrhea cases with rotavirus/norovirus GI infection, and four cases with rotavirus/norovirus GII.
Almost above of 60% gastroenteritis virus positive were collected from children under age 10, there were 60%, 88.8%, 81.8% and 67.7% positive for rotavirus, adenovirus, norovirus GI, norovirus GII, respectively. According to the ANOVA test method, it showed that there were strong statistic relationship between virus infection and patient age. However there was no significant difference for virus infectious frequency with geographical region.
The seasonal trends of norovirus detection were characterized by a peak incidence during spring (31.8 %), whereas adenovirus and rotavirus diarrhea occurred with summer and autumn, respectively. An epidemic of acute gastroenteritis was also identified as peaking mainly in July-September (>15 cases per month).
The purpose of this study is to develop one reliable detection method by applying molecular biology method for the gastroenteritis. In this study, we combined RT-nested-PCR and molecular epidemiological method to detect and identify total 270 suspected gastroenteritis cases. The result showed that Seminest -PCR method were more efficiently to detect these three enteric virus. According to the result of analysis of acute gastroenteritis cases, there were strong significant relationship between gastroenteritis cases and virus infectious frequency (ANOVA test, p<0.05).
Total 107 clinical samples of gastroenteritis virus from 2005 July to 2007 July were analyzed by sequence analysis and then identify serotype. The result showed that there were rotavirus G1, G3, G9 serotype ; Adenovirus type 2, 3, 41 ; Norovirus GI/8 and GII/4 detected in this study. In this study, it appeared that Taiwan epidemic gastroenteritis virus strains were closely related with other East Asian epidemic virus strains. It suggested that there were many epidemic gastroenteritis virus strains prevalent in East Asian region include Taiwan since 2005.
Key Words: Rotavirus, Adenovirus, Novovirus

並列關鍵字

Rotavirus ； Adenovirus ； Novovirus

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