Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66308
標題: 應用快速及高敏感之反轉錄恆溫環形核酸增幅法結合側流浸液試片偵測華氏囊病毒
Rapid and sensitive detection of infectious bursa disease virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick
作者: 蔡樹明
Tsai, Su-Ming
關鍵字: 傳染性華氏囊病;infectious bursa disease virus (IBDV);反轉錄恆溫環形核酸增幅法;側流浸液試片;reverse transcription loop-mediated isothermal amplification (RT-LAMP);lateral flow dipstick (LFD)
出版社: 微生物暨公共衛生學研究所
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摘要: 
傳染性華氏囊病(infectious bursal disease; IBD)為一種具高度傳染性的家禽疾病,廣泛流行於全世界之家禽業,感染IBDV之雞隻會有免疫抑制的現象,進而造成其他疾病的感染,造成嚴重的經濟損失。本次研究我們將發展反轉錄恆溫環形核酸增幅法(RT-LAMP)並結合FITC標定探針進行側流浸液試片(LFD)檢測傳染性華氏囊病毒,因過程具有3組特異性引子與特異性探針辨識才得以進行RT-LAMP-LFD反應,故具有高度特異性,不與雞隻自身核酸與其他病毒核酸進行非特異性反應,並且不需進行核酸電泳即可判讀結果,並且結合環狀區引子後可加快其反應速度約15分鐘,使其檢測過程可低於75分鐘,而RT-LAMP-LFD檢測極限可達0.1 PFU,是反轉錄聚合酶鏈反應 (RT-PCR) 的10000倍,巢氏反轉錄聚合酶鏈反應 (nested RT-PCR) 的10倍,具有高敏感度,在20個臨床樣本分別以RT-PCR 、nested RT-PCR、RT-LAMP與RT-LAMP-LFD四種方法進行檢測,其陽性率分別為20%、50%、60%與60%,並且在RT-PCR與nested RT-PCR之陽性樣本在RT-LAMP與RT-LAMP-LFD同樣為陽性,因此RT-LAMP-LFD用於檢測IBDV臨床樣本上,其檢出率是高於RT-PCR與nested RT-PCR的。因為RT-LAMP-LFD具有上述之快速性、便利性、特異性與高敏感性,故適合用於大規扶檢疫時偏遠的田野現場,並且可避免檢體運送時交叉感染其他雞場的可能性。

Infectious bursal disease (IBDV), an immunosuppressive disease for all aged chickens, still causes higher loss in the poultry industry years. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with a chromatographic lateral flow dipstick (LFD) for the detection of IBDV viruses was developed. The whole process can be completed in less than 75 minutes using biotin-labeled primers, an FITC-labeled DNA probe, and a LFD. The detection limit of IBDV using RT-LAMP-LFD was 10,000 fold and 10 fold higher than those of RT-PCR and nested RT-PCR, respectively. When other unrelated viruses and cells were tested, false positive results were not observed. In addition, the amplification efficiency of RT-LAMP was enhanced using loop primers and the RT-LAMP-LFD product can be detected as early as 15 minutes. Clinical samples were used to compare RT-PCR, nested RT-PCR, RT-LAMP, and the RT-LAMP-LFD assay and the positive rates were 20%, 50%, 60%, and 60%, respectively. In conclusion, this assay is an easy, rapid, accurate, and sensitive method for detecting IBDV and will provide greatly benefit to screen of field samples for veterinarians with poor resources.
URI: http://hdl.handle.net/11455/66308
其他識別: U0005-2001201115262800
Appears in Collections:微生物暨公共衛生學研究所

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