以分子生物學法進行台灣地區環境水體腸病毒之研究

THE STUDY OF MOLECULAR BIOLOGY METHODS FOR THE ENTEROVIRUSES IN WATER OF THE TAIWAN

萬民濤(Min-Tao Wan)

指導教授 ： 陳建先

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

台灣自1998年爆發腸病毒大流行以來，已成為每年流行於孩童的主要傳染病之一，由於腸病毒主要經由糞口傳播因此有必要就水環境進行調查與監測，然而迄今仍未有研究對台灣地區的水環境的腸病毒進行完整調查。本論文之研究目的即為利用分子生物學法建立一套可適用於環境病毒之監測流程並進行2003年至2005年台灣水環境的腸病毒調查，及探討環境因子對於腸病毒分佈之影響並以生物資訊學的方法探討腸病毒在台灣地區演化上的意義。
本研究以Real-time PCR及MPN-RT-PCR兩種方法比較不同電性濾膜搭配兩種流洗法共六種的病毒濃縮過濾之技術，發現以帶微量負電硝酸纖微濾膜(HA membrane)搭配H2SO4 / NaOH流洗法對於水中腸病毒有最佳的回收效率(100.0 %)。以兩組不同基因(5’UTR與VP1)及三組市售之病毒核酸萃取試劑比較不同形態樣本對於引子的萃取效果的分析，結果顯示以5’UTR在環境樣本的檢出率最好100.0 %(18/18)，而萃取試劑比較不論是萃取效率或是最低偵測極限均以Geneaid所生的病毒萃取試劑的66.7%的檢出率與5.3 ×10-4 MPN/ mL的最低偵測極限為佳。
本研究以RT-PCR、Seminest-PCR / Nest-PCR進行台灣地區的各類水體之檢測的結果顯示，台灣地區40.7 %(46/113)的環境水體中有存在腸病毒，顯示台灣地區的水環境確實有遭受腸病毒污染之虞。環境因子以水溫與腸球菌群對於腸病毒在淡水河潮間帶存在有與否有高度的關係。另外本研究將病毒以核酸定序分析後發現腸病毒71型、柯薩奇病毒A2型、A6型、伊科病毒11型及豬腸病毒9型分布在台灣各類水體環境，又經由分子演化分析後顯示與台灣2005的臨床病毒株在來源上具有一致性，此顯示了台灣地區的腸病毒疫情確實與水環境的傳播有一定程度的關連。

關鍵字

腸病毒 環境水體

並列摘要

There are large outbreaks of hand-foot-and-mouth disease (HFMD) occurred in Taiwan since 1998. Enteroviruses generally spread through the fecal-oral route, but they may cause the extensive infection via water. The aim of this study is to developed one reliable recovery method for enteroviruses from environmental water and to know the distribution of enteroviruses in the water in Taiwan between October 2003 to December 2005.
Aliquots of water samples were concentrated using nitrocellulose filters (HA membrane) with H2SO4/ NaOH is more highly recovery. 5’UTR-specific primer appeared more specific and sensitive than VP1-primer either in clinical or in environmental samples. The efficiencies of three virus RNA extraction methods in water samples were best to the Geneaid method. And the Geneaid kit resulted in the most consistently positive results at the lowest detection limit of enterovirus ( 5.3x10-4 MPN/ml ) in environmental water. Besides, the Geneaid extraction kit was faster and more cheaper than QIAgene and VIOGENE extraction kit.
We investigated total 113 water samples, including water treatment plants water ( raw water and treated water ), small water systems, hot spring, coastal water, river and hospital sewage . The identification methods used in this study includes reverse transcription (RT)-PCR, Seminest-PCR / Nest-PCR, and nucleotide sequence analysis of amplicons from 5' untranslated regions (5' UTR) genome.
Our results show that 40.7 % (46/113) present positive for enteroviruses in environmental water samples. It is proved that enteroviruses are widely distributed in Taiwan environment water. Besides, in environmental factor, the occurrence of enteroviruses was significantly related to temperature at ≦24.3 ℃ and enterococcus concentration in costal water and river (χ2 test; P < 0.05), but there was no significant related to pH, conductivity, salinity and HPC concentration in coastal water and river .(χ2 test; P > 0.05). In hot spring, there was no significant related to all environmental factor. The nucleotide sequence analysis of 5' UTR genome revealed that most isolates belonged to coxsackievirus A2(CA2), coxsackievirus A6(CA6),Enterovirus 71(EV71), Echovirus 11(ECV11) and procine-EV9, and that we observed Coxsackie A2, Coxsackie A6, EV71 and ECV11 were widely distributed and circulate between environmental and clinical in Taiwan.

並列關鍵字

Enteroviruses Water

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