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題 名 | 核酸偵測方法於草蝦白點病毒診斷上之應用=Application of Nucleic Acid Detection Methods in the Diagnosis of White Spot Baculovirus |
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作 者 | 黃千衿; 賴維川; 張菁雯; 羅竹芳; 簡茂盛; | 書刊名 | 臺灣畜牧獸醫學會會報 |
卷 期 | 68:3/4 民87.12 |
頁 次 | 頁103-111 |
分類號 | 439.66 |
關鍵詞 | 草蝦白點病毒; 聚合酵素連鎖反應; 核酸探針; 點墨雜交反應; White spot baculovirus; Polymerase chain reaction; Oligonucleotide probe; Dot blot hybridization; |
語 文 | 中文(Chinese) |
中文摘要 | 草 蝦 白 點 病 毒 (white spot baculovirus ; WSBV)屬 於動 物 病 毒中的桿狀病毒科 (Baculoviridae) 之桿狀病毒屬 (Baculovirus), 為目前養殖蝦類病毒 性疾病的重要病原, 並且造成蝦類的高死亡率。 本實驗即針對草蝦白點病毒之基因體 (genome) 中一段長約 4.6 kb 之 Sal I 限制 酵 素(restriction enzyme)切 割 DNA 片段進行 DNA 序列分析 (sequence analysis) , 並 進 而 設 計 出 進行聚合酵素連鎖反應 (polymerase chain reaction;PCR) 所需之引子 (primer) 與特異 性核酸檢驗探針 (DNA probe )。此完整 DNA 片段共含有 4,659 個核甘酸 (nucleotides) ,經由電腦 GCG program 分析此核酸序列顯示具有 6 個 開 讀框(open reading frame; ORF) 分別為:ORF 1: l~282、ORF 2: 291 ∼ 1493、 ORF 3: 2073~1750 (reverse)、ORF 4: 2360 ∼ 2617、ORF 5: 2971 ∼ 3867 及 ORF 6: 3892∼4284 。 而 PCR 反應中所使用之外側引子對 AA` 可增幅核甘酸序號 743 至 3617 長 2320 bp 之 DNA 片段, 而內側引子對 BB` 可增幅核甘酸序號 1039 至 3559 長 2874 bp 之 DNA 片段 。 使用外側引子對 AA` 並以病蝦 DNA 作為模版進行 PCR 後,經由瓊膠電泳分析顯示並無 產物出現; 然而再以內側引子對 BB` 進行巢式 PCR (nested-PCR) 後, 即可明顯見到大小 約 2.3 kb 之 PCR 產物產生。此外,草蝦白點病毒特異性核酸探針為針對核甘酸序號 2294 至 2323 長 30 mer 之寡核甘酸 (oligonucleotide), 經以 Digoxigenin (DIG) 標識後, 應用點墨雜交反應 (dot blot hybridization) 進行測試之結果顯示此探針具有相當的 靈 敏度,樣本中若含有 100 ng 的 WSBV DNA 即可被偵測出來; 而此探針在病蝦 DN 本中得到 陽性反應," 但在健康蝦則無,顯示此探針之專一性。 |
英文摘要 | White spot baculovirus (WSBV) belongs to the genus Baculovints of the family Baculoviridae. Currently, this virus is the most commonly found virus on shrimp farms and causes severe mortality in Asia and worldwide. In the past, diagnosis for WSBV was based on clinical signs and histopathological observation ; however, these methods often result in the misdiagnosis. Recently, the advance of molecular biological techniques has offered another rapid method for diagnosis of shrimp diseases with higher specificity and sensitivity. An accurate diagnosis and early prevention for WSBV should assist to minimize the mortality rate and reduce the economic losses. In this study, a Sal I restriction fragment approximately 4.6 kb in size of the WSBV genomic DNA was cloned and completely sequenced for nucleotide sequence analysis. The 4,659 nucleotides were further analyzed by the computer GCG program and revealed six open reading frames--ORF 1: 1-282, ORF 2: 291-1493, ORF 3: 2073-1750 (reverse), ORF 4: 2360-2617, ORF 5: 2971-3867, ORF 6: 3892-4284, respectively. Two primer pairs (AA` and BB`) were designed according to the nucleotide sequence of this particular DNA fragment and were subjected to polymerase chain reaction (PCR) experiments. PCR with outer primer pair AA` followed by a nested-PCR with primer pair BB` could amplify a specific DNA fragment to WSBV genomic DNA approximately 2.3 kb in size. Furthermore, a 30-mer oligonucleotide probe specific to this WSBV genomic DNA fragment was labeled using DIG labeling system and used to detect the presence of WSBV DNA in the infected shrimp by dot blot bhbridization. The results demonstrated that as little as little as 100 ng WSBV DNA could be detected in the infected shrimp but not in healthy shrimp DNA, confirming the specificity and sensitivity of this probe. |
本系統中英文摘要資訊取自各篇刊載內容。