Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30779
標題: 利用即時定量聚合酶鏈鎖反應技術偵測日本腦炎病毒
Using the real-time quantitative polymerase chain reaction to detect Japanese encephalitis virus
作者: 盧慧真
Lu, Hui-Zhen
關鍵字: 即時定量聚合酶鏈鎖反應;real-time quantitative polymerase chain reaction;日本腦炎病毒;Japanese encephalitis virus
出版社: 昆蟲學系所
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摘要: 
日本腦炎(Japanese encephalitis)是流行於亞洲地區的人畜共通疾病,對人及動物的生命造成威脅。台灣原是基因三型日本腦炎病毒的主要流行區,而在2008年自病媒蚊體分離出日本腦炎病毒基因一型後,田間的日本腦炎病毒轉為基因一型的流行地區。本試驗以日本腦炎病毒RNA基因體中負責轉譯、具有RNA helicase 活性的NS3蛋白核酸序列設計real-time qPCR引子對,測得real-time qPCR日本腦炎病毒基因一型的引子(G1-3)偵測極限為 2×101 copies/μl,基因三型的引子(G3-3)的偵測極限為 2×102 copies/μl;建立具快速、靈敏、可以定量、不受蚊體核酸序列干擾等優點的即時定量聚合酶鏈鎖反應(real-time quantitative polymerase chain reaction, real-time qPCR)技術。並利用此技術探討三斑家蚊(Culex tritaeniorhynchus)在吸食感染基因一型日本腦炎病毒(YL2009-4)之後,分別在20°C、25°C、30°C等不同溫度條件飼養,連續14天觀察病毒量之變化。結果顯示,三斑家蚊雌蟲吸食感染基因一型病毒,於20°C下,一週後蚊體內的病毒量,為吸食病毒血混合液後蚊體的2.834倍,25°C下為23.132倍,30°C下為0.002倍;兩週後,20°C下為0.949倍,25°C下為1,255.994倍,30°C下為145.213倍;而在基因三型病毒的結果,25°C下,一週後蚊體內的病毒量為0.009倍;兩週後為0.078倍。此外,比較在25°C溫度條件下日本腦炎病毒基因一型(YL2009-4)與基因三型(T1P1)在三斑家蚊蚊體之複製情形,結果顯示三斑家蚊對日本腦炎病毒基因一型與基因三型感受性具有差異。

Japanese encephalitis is the prevalent epizootic disease in Asia area that threatens human and animal health. Japanese encephalitis virus (JEV) genotype 3 was endemic in Taiwan. JEV genotype 1 was first isolated from mosquito pools in Taiwan in 2008. Since then, JEV genotype 3 has switched to genotype 1 in Taiwan. In this study, real-time qPCR primer pairs were designed according JEV NS3 gene encoding RNA helicase. It revealed that the detection limits of real-time qPCR for primers G1-3 of JEV genotype 1 and primer G3-3 of genotype 3 were 2×101 copies/μl and 2×102 copies/μl, respectively. To establish a rapid, sensitive, quantitative and specific real-time quantitative polymerase chain reaction (real-time qPCR) is also the main purpose in our study. The real-time qPCR assay was used to detect viral RNA in these Culex tritaeniorhynchus infected with JEV genotype 1, YL2009-4, via oral feeding during the extrinsic incubation period of 0 to 14 days at 20°C, 25°C, and 30°C, respectively. The results revealed that the JEV viral loads of mosquitoes infected YL2009-4 after a week were 2.834 fold at 20°C, 23.132 fold at 25°C, and 0.002 fold at 30°C; after 2 weeks the viral loads were 0.949 fold at 20°C, 1,255.994 fold at 25°C, and 145.213 fold at 30°C compared with the mosquitoes ingestion viral loads. However, it revealed that JEV viral loads of mosquitoes infected T1P1 after a week were 0.009 fold, after 2 weeks were 0.078 fold at 25°C. In addition, comparing the replication of JEV genotype 1, YL2009-4, and JEV genotype 3, T1P1, in Cx. tritaeniorhynchus at 25°C, it revealed that Cx. tritaeniorhynchus showed different susceptibilities to JEV genotype 1 and genotype 3.
URI: http://hdl.handle.net/11455/30779
其他識別: U0005-2207201316074900
Appears in Collections:昆蟲學系

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