本文選題：牛傳染性鼻氣管炎 + gB、gE基因　； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：本研究根據(jù)GenBank中登錄的IBRV gE和gB基因序列,設(shè)計(jì)2對(duì)特異性引物及Taqman探針,建立了牛傳染性鼻氣管炎病毒(IBRV)的雙重?zé)晒舛縋CR檢測(cè)方法并進(jìn)行了敏感性、特異性及臨床樣本的檢測(cè)。結(jié)果表明,該方法敏感性高,最低可檢測(cè)約2拷貝/μL;特異性強(qiáng),對(duì)牛支原體、牛副流感病毒3型、牛病毒性腹瀉病毒、巴氏桿菌、牛、羊布魯氏菌的檢測(cè)結(jié)果均為陰性。重復(fù)性試驗(yàn)中,組內(nèi)和組間變異系數(shù)均小于2%。利用建立的方法對(duì)54份牛肺和18份牛鼻拭子樣品進(jìn)行檢測(cè),結(jié)果54份牛肺中IBRV陽性17份,其中雙陽性12份,gB陽性而gE陰性5份,雙陰性37份;18份鼻拭子中IBRVgB陽性而gE陰性1份,雙陰性17份。研究表明所建立的熒光定量PCR檢測(cè)方法具有快速、敏感、準(zhǔn)確等優(yōu)點(diǎn),可以用于IBRV的檢測(cè)。此外,本試驗(yàn)根據(jù)GenBank發(fā)表的IBRVgB和gE基因序列,設(shè)計(jì)合成2對(duì)帶有酶切位點(diǎn)的引物,構(gòu)建了用于真核表達(dá)的載體。結(jié)果表明,本試驗(yàn)成功擴(kuò)增出目的片段,并將其克隆到pMD-19Tsimple載體。將鑒定成功的pMD-19Tsimple-gB、pMD-19Tsimple-gE重組質(zhì)粒經(jīng)BamH Ⅰ和EcoR Ⅰ雙酶切后,連接到表達(dá)載體pFastBacl,得到了重組質(zhì)粒pFBgB、pFBgE。經(jīng)一系列的鑒定,得到了含gB、gE基因的重組桿粒,為轉(zhuǎn)染昆蟲細(xì)胞(Sf9/Sf21)奠定了基礎(chǔ)。
[Abstract]:Based on the sequence of IBRV GE and GB genes registered in GenBank, two pairs of specific primers and Taqman probes were designed to detect bovine infectious rhinotracheitis virus (IBRV) by double fluorescence quantitative PCR. Detection of specificity and clinical samples. The results showed that the method was highly sensitive and could be detected at least 2 copies / 渭 L, and had strong specificity, negative for Mycoplasma bovis, bovine parainfluenza virus type 3, bovine viral diarrhea virus, Pasteurella spp, bovine and sheep brucella. In the repeatability test, the coefficient of variation within and between groups was less than 2%. The established method was used to detect 54 bovine lungs and 18 bovine nasal swabs. The results showed that 17 of 54 bovine lung samples were positive for IBRV, of which 12 were positive for IBRV and 5 were negative for GE, and 37 were positive for IBRVgB and 1 for GE in 18 nasal swabs. 17 cases were double negative. The results show that the established fluorescence quantitative PCR method has the advantages of fast, sensitive and accurate, and can be used for the detection of IBRV. In addition, based on the IBRVgB and GE gene sequences published by GenBank, two pairs of primers with restriction endonuclease sites were designed and synthesized to construct the eukaryotic expression vector. The results showed that the target fragment was successfully amplified and cloned into pMD-19Tsimple vector. The identified recombinant plasmid pMD-19Tsimple-gE was digested by BamH 鈪，

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