本文選題：反轉(zhuǎn)錄環(huán)介導(dǎo)等溫擴增(RT-LAMP) + H亞型禽流感病毒(H-AIV)��； 參考：《農(nóng)業(yè)生物技術(shù)學(xué)報》2016年09期

【摘要】：本研究利用反轉(zhuǎn)錄環(huán)介導(dǎo)等溫擴增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技術(shù)建立了H5亞型禽流感病毒(Avian influenza virus,AIV)檢測方法。首先,針對H5亞型禽流感病毒血凝素(hemagglutinin,HA)基因8個保守區(qū)域設(shè)計了3對擴增引物。然后,添加到RT-LAMP反應(yīng)體系中,并加入優(yōu)選的凍干保護劑進行冷凍干燥。取等溫擴增凍干試劑,用雙蒸水溶解并恢復(fù)至原體積后,在63℃反應(yīng)1 h,可檢出102拷貝的目的基因,比RT-PCR方法敏感約10倍,并且證明冷凍干燥對等溫擴增反應(yīng)敏感性沒有影響。另外,利用等溫擴增凍干試劑對禽流感病毒、新城疫病毒(Newcastle disease virus,NDV)和傳染性支氣管炎病毒(Infectious bronchitis virus,IBV)進行檢測,結(jié)果顯示特異性良好。為了便于臨床應(yīng)用,在反應(yīng)結(jié)束后加入由SYBR Green I與羥基萘酚藍(hydroxynaphthol blue,HNB)組成的顯色液,可通過肉眼觀察顏色變化判定RT-LAMP結(jié)果,與瓊脂糖凝膠電泳方法判定結(jié)果一致。為考察等溫擴增凍干試劑的臨床應(yīng)用效果,對326份臨床樣品進行了檢測。結(jié)果表明,RT-LAMP與RT-PCR檢出陽性樣本數(shù)分別為38份和31份,表明RT-LAMP方法陽性檢出率高于RT-PCR方法。因此,本研究建立的H5亞型流感病毒RT-LAMP反應(yīng)體系,可以凍干處理并經(jīng)顯色觀察結(jié)果,便于研制成試劑盒產(chǎn)品,其敏感性高、特異性好、操作簡捷,利于在基層獸醫(yī)實驗室和養(yǎng)殖場推廣應(yīng)用。
[Abstract]:In this study, a reverse transcription loop-mediated isothermal amplification assay (RT-LAMPP) technique was used to detect avian influenza virus (Avian influenza virus) of H5 subtype. Firstly, three pairs of primers were designed for 8 conserved regions of hemagglutinin (H5) gene of H5 subtype avian influenza virus. Then, it was added to RT-LAMP reaction system and freeze-dried protective agent was added. Isothermal amplification freeze-dried reagent was extracted, dissolved and restored to its original volume with double distilled water. After 1 hour reaction at 63 鈩，

本文編號：1964133