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- A Visual DNA Chip for Simultaneous Detection, Genotyping, and Differentiation of Wild-type and Vaccine-type Classical Swine Fever Virus
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題 名 | A Visual DNA Chip for Simultaneous Detection, Genotyping and Differentiation of Wild-type and Vaccine-type Classical Swine Fever Virus=以目視DNA晶片同時檢測、分型及區別豬瘟野外毒及疫苗毒 |
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作 者 | 潘居祥; 顧有為; 鍾明華; 趙磐華; 賴秀穗; | 書刊名 | 臺灣獸醫學雜誌 |
卷 期 | 34:2 2008.06[民97.06] |
頁 次 | 頁66-76 |
分類號 | 437.246 |
關鍵詞 | 豬瘟病毒; DNA晶片; 探針; 反轉錄聚合酶鏈反應; Classical swine fever virus; DNA chip; Probe; RT-PCR; |
語 文 | 英文(English) |
中文摘要 | 應用反轉錄聚合酶鏈反應(Reverse transcription-polymerase chain reaction; RT-PCR)增幅後再進行核酸定序是目前檢測野外豬瘟並排除疫苗毒幹擾實驗室診斷之主要方法。本論文為了同時檢測,分型及區別豬瘟野外毒及疫苗毒因而發展目視DNA晶片檢測法。豬瘟病毒特異性引子及探針係依據病毒基因3端未轉譯區核酸序列而設計,採用生物素標識引子進行單步驟RT-PCR反應,隨後與固定在高分子塑膠晶片上的探針進行雜合反應(Hybridization)。本方法可精確地將豬瘟病毒區分為三種主要基因型,並可同時區別野外毒及疫苗毒。傳統RT-PCR 方法可檢測豬瘟野外毒之最低力價為10 TCID50/mL,DNA晶片可檢測之最低病毒力價為1 TCID50/mL,DNA晶片相較於RT-PCR方法敏感性高10倍。RT-PCR 結合DNA探針雜合技術可提高檢測敏感性,可快速鑑定臨床檢體中豬瘟病毒的基因型別,並區別野外毒及疫苗毒。 |
英文摘要 | Routinely, RT-PCR followed by DNA sequencing has been the method used to detect classical swine fever virus (CSFV) and exclude the interference of vaccine viruses for the field cases. Here, a DNA chip assay was developed to enable simultaneous detection, genotyping and differentiation between wild and vaccine-type CSFV. Specific oligonucleotide primers and probes were designed in the 3'non-translated region of the CSFV genome. One-step RT-PCR amplification was performed with biotin-labeled primers, followed by hybridization to the DNA probe immobilized on the plastic chip. The DNA chip can not only accurately differentiate between the three major genotypes of CSFV, but can also discriminate between wild-type and vaccine-type CSFV. The limit of detection for wild-type virus was 10 TCID50/mL for RT-PCR and 1 TCID50/mL for the DNA chip. The sensitivity of the visual DNA Chip was 10 times higher than that of the RT-PCR confirmed by agarose gel. We conclude that RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping CSFV and discriminating between wild-type and vaccine-type CSFV in clinical samples. |
本系統中英文摘要資訊取自各篇刊載內容。