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Studies on the Entry and Distribution of Aquatic Birnavirus in Tilapia (Tilapia aureas) by the Single-Tube Reverse trnascription-Polymerase Chain Reaction Technique
|關鍵字:||Single-Tube Reverse trnascription-|
Polymerase Chain Reaction
single-tube reverse transcription polymerase chain reaction；
醣核酸病毒（aquatic birnavirus）進入吳郭魚（Tilapia aureus）體內
）及聚合酵素連鎖反應（polymerase chain reaction）分開，使得二個
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
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.
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