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標題: 利用反轉錄-環圈式恆溫核酸增幅法快速偵測傳染性華氏囊病病毒
Development of reverse transcription loop-mediated isothermal amplification method for rapid detection of infectious bursal disease virus
作者: 林逸秋
Lin, Yi-Chiu
關鍵字: IBDV;傳染性華氏囊病;LAMP;病毒;恆溫擴增法
出版社: 生物科技學研究所
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傳染性華氏囊病(infectious bursal disease, IBD) 為一高度傳染性疾病,主要流行年輕雞隻,其病原為傳染性華氏囊病病毒 (infectious bursal disease virus, IBDV)。環圈式恆溫核酸增幅法 (loop-mediated isothermal amplification, LAMP) 則為一簡便、快速、專一之新式核酸增幅方法,利用特殊設計之引子加上鏈取代型 DNA 合成酶之特性,可在恆溫環境下快速合成大量目標核酸序列。本篇選用 LAMP 法並結合反轉錄作用,發展一偵測華氏囊檢體中之 IBDV 之方法。實驗中所使用之引子來自 IBDV 外鞘蛋白 VP2 之基因片段,首先經 VP2 cDNA 測試可正確辨認目標核酸片段,再萃取 IBDV RNA測試RT-LAMP之敏感度,得其偵測極限可達 0.01 fg 目標核酸。此結果相較於一般 PCR 為佳。且其具專一性,對非目標核酸的雞貧血病毒、禽類流行性感冒病毒等基因皆無反應。實際抽取染病華氏囊檢體中之 RNA,經反轉錄後亦可以得到 LAMP 反應,顯示此方法可檢測出檢體中的傳染性華氏囊病病毒。且其可同時進行反轉錄及恆溫擴增 ,比一般RT-PCR 來得簡單且快速。實驗結果顯示,RT-LAMP 可運用於IBDV 之診斷。

Infectius bursa disease (IBD) is a highly contagious disease in young chicken. The pathogen of IBD is infectious bursal disease virus (IBDV). Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method with advantages of simplicity, rapidity, and specificity. The amplification depends on a set of special primer combined with a DNA polymerase with strand replacement activity, and large amounts of products were synthesized at isothermal condition in one hour. In this study, LAMP combined with reverse transcription for detection of IBDV was performed. The initial assay confirmed the primer set amplified correct target. Extracted genomic RNA of IBDV was used for the sensitivity test of RT-LAMP. The limit of detection was determined to be 0.01 fg RNA of IBDV in the present study. In the specificity test, LAMP only amplified sample contained IBDV; CAV and AIV were negative results. In the RT-LAMP assay with clinical samples, the IBDV in bursa samples detected correctly. Moreover, the LAMP method and reverse transcription worked at the same time that more convenient than conventional RT-PCR. These data suggest the RT-LAMP method is a potential diagnosis tool for detecting IBDV.
其他識別: U0005-2108200918365600
Appears in Collections:生物科技學研究所

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