發(fā)布時間：2018-06-16 15:22

  本文選題：兔出血癥病毒 + RT-LAMP　； 參考：《中國獸醫(yī)科學(xué)》2017年06期

【摘要】：為建立一種可視化的兔出血癥病毒RT-LAMP檢測方法,根據(jù)兔出血癥病毒(rabbit hemorrhagic disease virus,RHDV)的主要結(jié)構(gòu)蛋白VP60基因的序列設(shè)計合成4條特異性引物,以重組pMD19-T-VP60質(zhì)粒為模板,通過反應(yīng)條件與反應(yīng)體系優(yōu)化,建立RHDV的環(huán)介導(dǎo)等溫擴增(LAMP)檢測方法,測定RT-LAMP對RHDV的最低檢測限,并與普通RT-PCR進行比較。結(jié)果顯示,該RT-LAMP的最佳反應(yīng)溫度為62℃,反應(yīng)時間為40 min,對RHDV的最低檢測限為50 copies/L,常規(guī)RT-PCR的檢測限為5×10~3copies/L,表明建立的RT-LAMP的靈敏度顯著高于常規(guī)RT-PCR。建立的RT-LAMP的全部反應(yīng)可在1 h內(nèi)完成;反應(yīng)結(jié)束后加入熒光染料Et Br可肉眼判定粉色為陽性,而棕色則為陰性,且與兔巴氏桿菌、大腸桿菌、沙門菌、產(chǎn)氣莢膜梭菌、無乳鏈球菌及肺炎克雷伯菌無交叉反應(yīng)。上述結(jié)果表明,建立的RT-LAMP簡便、快速、靈敏、特異,且不需昂貴儀器設(shè)備,適合在基層推廣應(yīng)用。
[Abstract]:In order to establish a visual detection method for rabbit haemorrhagic disease virus RT-lamp, four specific primers were designed and synthesized according to the sequence of the main structural protein VP60 of rabbit hemorrhagic disease virus. The recombinant plasmid pMD19-T-VP60 was used as template. Based on the optimization of reaction conditions and reaction system, a method for detection of RHDV by ring mediated isothermal amplification (Lampp) was established, and the minimum detection limit of RT-LAMP for RHDV was determined and compared with that of common RT-PCR. The results showed that the optimum reaction temperature of RT-LAMP was 62 鈩，

本文編號：2027153