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標題: 應用聚合酵素連鎖反應檢測水生兩段雙股核醣核酸病毒
Developing the Polymerase Chain Reaction Technique to Detect Aquatic Birnavirus
作者: 李建賢
Lee, Jiann Shyan
關鍵字: PCR
Aquatic Birnavirus
出版社: 獸醫學系
摘要: 本實驗之主要目的,在於利用聚合酵素連鎖反應(PCR)的方法檢測水生兩 段雙股核醣核酸病毒。實驗總共使用七組引子(A組~G組),來檢測五株已 a、WB、Sp、Ab、VR299,以及三株台灣分離株3372、MFK、 CV-HB-1。由 實驗結果得知,A組引子可以檢測Ja、WB、VR299三株病毒;B組引子可以 檢測Ja、WB、Sp、VR299四株病毒;C、D、F及G組引子對於八株待測病毒 均能檢測;而E組引子可以檢測Ja、WB、VR299、MFK、CV-HB-1五株病毒。 使用此七組引子進行PCR檢測上述之病毒,均只有一個主要產物出現,並 且均可以與IBDV及IHNV作區別診斷,因此其特異性非常高。由實驗結果顯 示,所開發之方法的靈敏度為15fg∼15pg病毒RNA(約相當於2×10^2∼2 ×10^5個病毒顆粒)。本實驗進一步嘗試開發單一試管反轉錄聚合酵素連 鎖反應(single- tube RT-PCR)診斷技術,由實驗結果顯示,所開發的單 一試管RT-PCR診斷技術能直接由魚的組織中檢測出水生兩段雙股核醣核酸 病毒,並且整個檢測過程僅需12個小時即可完成檢測工作,因此比傳統之 PCR方法能更快速、更準確並且更具敏感地檢測出水生兩段雙股核醣核酸 病毒。
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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