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Developing the Polymerase Chain Reaction Technique to Detect Aquatic Birnavirus
Lee, Jiann Shyan
鎖反應(single- tube RT-PCR)診斷技術，由實驗結果顯示，所開發的單
The aim of this study was to development a rapid, sensitive, and highly specific detection method for aquatic birnaviruses by the polymerase chain reaction (PCR). 7 sets of primer (primer set A~G) were selected from the major capsid polypeptide VP2 gene of aquatic birnaviruses. 8 strains of viruses including Ja, WB, Sp, Ab, VR299, 3372, MFK, and CV-HB-1 were used in this study. The viruses (3372, MFK and CV-HB-1) were isolated in Taiwan. The result showed that Ja, WB and VR299 were able to be detected by the primer set A. Ja, WB, Sp and VR299 could be detected by the primer set B. The primer set C, D, F, and G could detect all of the 8 viral strains. The primer set E could detect Ja, WB, VR299, MFK and CV-HB-1. Each primer set only amplified one major product from tested aquatic birnaviruses but not infectious hematopoietic necrosis virus (IHNV), infectious bursal disease virus (IBDV), and uninfected CHSE-214 cells. The results revealed that sensitivity of the developed method was as little as 15 fg to 15 pg of purified viral dsRNA when amplification product was visualized by ethidium bromide-stained gel. Furthermore, an assay protocol base on single-tube reverse transcription-PCR (RT-PCR) for the detection of aquatic birnaviruses using wax to divide reverse transcriptase and Taq polymerase has been developed. This technique could detect aquatic birnaviruses directly from fish tissue. The advantage of this technique was to avoid viral propagation using cell culture technique. All the procedures of RT-PCR detection could be done in 12 hours. The single-tube RT- PCR seems to be a quick senstive and specific viable alternative tool for detecting aquatic birnaviruses.
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