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題名 | 應用反轉錄恆溫環形核酸增幅法偵測豬瘟病毒=Detection of Classic Swine Fever Virus by Application of Reverse Transcriptase Loop-mediated Isothermal Amplification Method |
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作者 | 鄧明中; 林育如; 宣詩玲; 李敏旭; 黃天祥; 黃金城; 簡茂盛; Deng, Ming-chung; Lin, Yu-ju; Hsuan, Shih-ling; Lee, Ming-shiuh; Huang, Tien-shine; Huang, Chin Cheng; Chien, Maw-sheng; |
期刊 | 臺灣獸醫學雜誌 |
出版日期 | 20080600 |
卷期 | 34:2 2008.06[民97.06] |
頁次 | 頁97-105 |
分類號 | 437.246 |
語文 | chi |
關鍵詞 | 豬瘟病毒; 反轉錄恆溫環形核酸增幅法; 聚合酵素鏈鎖反應; 兔化豬瘟; Classical swine fever virus; Reverse transcriptase loop-mediated isothermal amplification; Polymerase chain reaction; Lapinized hog cholera; |
中文摘要 | 由於聚台酵素連鎖反應(Polymerase chain reaction, PCR)能在短時間內增幅特定基因片段,因此常被應用於許多病原核酸之偵測與診斷。但一般的PCR需要精密的熱循環器(thermal cycler),對於田間或臨床上無此設備而又必須進行快速確診時,往往喪失先機而致疫情擴散。為克服此一狀況,許多方便可靠的核酸增幅技術被開發出來,其中又以恆溫環形核酸增幅法(loop-mediated isothermal amplification; LAMP)最廣為應用。由於此法所需設備較為簡便且操作容易,因此我們藉由此技術來開發快速偵測豬瘟病毒核酸的方法。為了確認此新技術之敏感性與特異性符合診斷之需求,我們與即時定量RT-PCR (real time RT-PCR)、巢式RT-PCR (nested RT-PCR)以及傳統RT-PCR進行比較,結果顯示RT-LAMP可偵測到約0.32 TCID50的病毒量,與real time RT-PCR、nested RT-PCR敏感度相似,遠比傳統RT-PCR敏感約1500倍,且RT-LAMP不會增幅與豬瘟病毒同屬的牛病毒性下痢病毒核酸,特異性非常高。因此,應用RT-LAMP核酸增幅法開發豬瘟病毒核酸的快速檢測技術,對於未來田間疫情的診斷將有莫大的幫助。 |
英文摘要 | Polymerase chain reaction (PCR) has been widely used to amplify specific nucleotide sequences from viral pathogens. However, performance of traditional PCR requires an expensive thermal cycler, which is inconvenient for application in field or in clinical practice. To improve the deficiency, a novel method based on PCR have been developed Loop-mediated isothermal amplification (LAMP) can be carried out in a low-priced thermal bath. We therefore developed the rapid diagnostic method based on LAMP to detect classical swine fever viruses. Viral RNA extracted from cell culture or infected samples was mixed with specific primers and DNA polymerase. After reacting in a 63℃ thermal cycler for one hour. The amplicons were analysed. Our results indicated that the sensitivity of RT-LAMP was equal to real time RT-PCR and nested RT-PCR, detecting the lowest viral titer detectable by the assay was about 0.32 TCID50. Based on this finding, we suggest that RT-LAMP can be used to detect the specific nucleotide sequences of classical swine fever virus from field infected samples. |
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