篇名:
以目視DNA晶片同時檢測,分型及區別豬瘟野外毒及疫苗毒A visual DNA chip for simultaneous detection, genotyping, and differentiation of wild-type and vaccine-type classical swine fever viru
作者:
潘居祥;顧有為;鍾明華;趙磐華;賴秀穗*
中文摘要:
應用反轉錄聚合?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干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 to exclude the interference of vaccine viruses in clinical samples. Here, a DNA chip assay was developed to enable simultaneous detection, genotyping and differentiation between wild-type 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 chips. The DNA chips not only can accurately differentiate three major CSFV genotypes, but also can 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 chips. The sensitivity of the visual DNA chip was 10 times higher than that of the RT-PCR confirmed by agarose gel electrophoresis. We conclude that RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping of CSFV and for discriminating between wild-type and vaccine-type CSFV in clinical samples.
備註:
年份:西元 2004 年
期別:第 40 期
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