Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13125
標題: 應用單一試管反轉錄聚合酵素連鎖反應技術研究水生兩段雙股核醣核酸病 毒進入吳郭魚之途徑及其在體內之分佈消長
Studies on the Entry and Distribution of Aquatic Birnavirus in Tilapia (Tilapia aureas) by the Single-Tube Reverse trnascription-Polymerase Chain Reaction Technique
作者: 魏雅伶
Wi, Yea-Lin
關鍵字: Single-Tube Reverse trnascription-
單一試管反轉錄聚合酵素連鎖反應
Polymerase Chain Reaction
quatic
Birnavirus
水生兩段雙股核醣核酸病毒
出版社: 獸醫學系
摘要: 中文摘要本研究之目的在建立及應用單一試管反轉錄聚合酵素連鎖反應( single-tube reverse transcription polymerase chain reaction; single-tube RT-PCR)技術並結合病毒分離技術,以研究水生兩段雙股核 醣核酸病毒(aquatic birnavirus)進入吳郭魚(Tilapia aureus)體內 的途徑及該病毒在吳郭魚體內分佈及消長情形,進而探討本病毒在魚類之 可能致病機轉。本研究嘗試以蠟將反轉錄作用(reverse transcription )及聚合酵素連鎖反應(polymerase chain reaction)分開,使得二個 反應所需的酵素不會相互干擾,並所有反應在單一試管內完成因而可減少 許多操作時間。由實驗結果顯示本技術具有良好特異性;對同病毒科之禽 類傳染性華氏囊炎病毒(IBDV)、魚類之傳染性造血組織壞死病毒(IHNV )、未感染病毒之CHSE-214細胞及未感染病毒之吳郭魚組織均不會產生特 定產物,僅對水生兩段雙股核醣核酸病毒產生特定產物。並且由試驗結果 顯示本技術具有良好的敏感性;即使病毒RNA僅為15 pg(約相當200個病 毒顆粒)時仍可測出病毒核酸之存在。以3372台灣分離株感染吳郭魚以研 究水生兩段雙股核醣核酸病毒在活體外(in vitro)及活體內(in vivo )病毒複製之情形。活體外感染實驗結果顯示不論是以病毒分離或是單一 試管反轉錄聚合酵素連鎖反應等方法均可在所有之感染組織(皮膚、鰭、 鰓、肝、腎、脾及肌肉)中檢測到該病毒之存在,其中以肌肉及皮膚之病 毒力價最高而鰓最低。並由感染組織中之病毒力價消長情形顯示於攻毒後 至攻毒後第三天所有感染組織之病毒力價並無顯著的變化。同時病毒亦可 在組織培養液中偵測到,由病毒力價之變化情形可顯示所有感染組織自攻 毒後至攻毒後第三天,病毒力價每天均有顯著的上升情形,其中以腎、肝 及肌肉之病毒力價最高而鰭最低。然而因為自攻毒後第四天起組織產生死 後變化,故無法進行病毒之分離測定,僅能以單一試管反轉錄聚合酵素連 鎖反應檢測病毒核酸是否存在。結果顯示在攻毒後第四天仍可由組織及其 培養液中偵測到病毒核酸的存在。由活體內感染試驗結果顯示以浸潤方式 感染魚隻時,病毒力價以腸道最高,並於攻毒後三天內仍可在鰓中偵測到 。如改口服方式感染魚隻時卻僅能在腸道檢測到病毒。以腹腔注射方式感 染魚隻,病毒於感染之後可立刻被偵測到;即病毒可藉由血液循環迅速地 散播至全身,並且病毒持續地出現於組織器官之中,可達35天之久,其中 以腸道能檢測到病毒之時間最長而鰓最短。病毒力價則以腸道、腎及脾最 高而鰓最低,並且所有之組織在攻毒後第一天病毒力價有逐漸上升的情形 且在第一週內達到最高峰,而後病毒力價則緩慢地下降。
AbstractThe aims of this investigation are to study the entry and distribution of aquatic birnavirus in tilapia (Tilapia aureus) by the single-tube reverse-transcription polymerase chain reaction (single tube RT-PCR) and virus isolation techniques. The principle of the developed method for the detection of aquatic birnavirus RNA in fish tissues is to combine both steps in one reaction tube with a wax interface which prevented the inhibitory effect of RTase on Taq polymerase.It could shorten the time of RT-PCR to separate the reverse transcription of viral RNA into cDNA and subsequent amplification of polymerase chain reacitons. The results of the specificity of the single-tube RT-PCR technique revealed that it was sensitive enough to differentiate aquatic birnavirus from infectious bursal disease virus (IBDV) belonged to the same Birnaviridae and infectious heamotopoietic necrosis virus (IHNV) caused a serve diseases in fish. The results of the sensitivity showed that our method could detect the viral amount of the RNA to 15 pg that is equal to about 200 viral particles.In order to understand the entry and distribution of aquatic birnavirus in fish, the Taiwan isolate, 3372, was used to infect tilapia in vitro and in vivo then the viral titers in organs and tissues from infected fish were studied. The results of in vitro infection revealed that existences of aquatic birnavirus were demonstrated in all collected organs and tissues including skin, fin, gill, liver, kidney, spleen, and muscle and in the tissue culture medium by the single-tube RT-PCR and virus isolation techniques. The highest titers were found in skin and muscle but gill had the lowest titer. There were no remarkable evolution in all inoculated organs and tissues during the experiment days. However, viral titers in the tissue culture medium significantly increased within everyday after infection. The lowest titer showed in fin but the highest titer were found in kidney, liver, and muscle. Because the tissues had post-mortem change in the 4th day post infection, the virus isolation technique was not suitable for virus detection. At this moment, our method showed that it is efficient to detect the viral RNA from the post- mortem changed tissues. The results of the in vivo infection experiment suggested that viruses could be recovered in intestine from infected fish by immersion. The viral titers could persistently detected in the organs up to the 3rd day post infection. Instead of infection by feeding, viruses could be found in intestine only. At the same situation, viruses were detected in most tissues from infected fish which were inoculated viruses by intraperitoneal injection; it suggested that viruses were spreaded rapidly by the circulatory system to all organs. Viruses were able to present in fish even at the 35th day after injection. The recovered time of the longest and the shortest were in intestine and in gill, respectedly. Intestine , kidney and spleen had the highest titers but the lowest titers was found in gill. After the 1st day post infection, the viral titers in all collected organs increased and proceeded up to the 7th day then they decreased.
URI: http://hdl.handle.net/11455/13125
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