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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;聚合酵素連鎖反應;Aquatic Birnavirus;IPNV;水生兩段雙股核醣核酸病毒;傳染性胰壞死病毒
出版社: 獸醫學系
a、WB、Sp、Ab、VR299,以及三株台灣分離株3372、MFK、 CV-HB-1。由
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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