IBRV双重荧光定量PCR检测方法的建立

发布时间：2018-05-25 02:18

  本文选题：牛传染性鼻气管炎 + gB、gE基因　； 参考：《内蒙古农业大学》2017年硕士论文

【摘要】：本研究根据GenBank中登录的IBRV gE和gB基因序列,设计2对特异性引物及Taqman探针,建立了牛传染性鼻气管炎病毒(IBRV)的双重荧光定量PCR检测方法并进行了敏感性、特异性及临床样本的检测。结果表明,该方法敏感性高,最低可检测约2拷贝/μL;特异性强,对牛支原体、牛副流感病毒3型、牛病毒性腹泻病毒、巴氏杆菌、牛、羊布鲁氏菌的检测结果均为阴性。重复性试验中,组内和组间变异系数均小于2%。利用建立的方法对54份牛肺和18份牛鼻拭子样品进行检测,结果54份牛肺中IBRV阳性17份,其中双阳性12份,gB阳性而gE阴性5份,双阴性37份;18份鼻拭子中IBRVgB阳性而gE阴性1份,双阴性17份。研究表明所建立的荧光定量PCR检测方法具有快速、敏感、准确等优点,可以用于IBRV的检测。此外,本试验根据GenBank发表的IBRVgB和gE基因序列,设计合成2对带有酶切位点的引物,构建了用于真核表达的载体。结果表明,本试验成功扩增出目的片段,并将其克隆到pMD-19Tsimple载体。将鉴定成功的pMD-19Tsimple-gB、pMD-19Tsimple-gE重组质粒经BamH Ⅰ和EcoR Ⅰ双酶切后,连接到表达载体pFastBacl,得到了重组质粒pFBgB、pFBgE。经一系列的鉴定,得到了含gB、gE基因的重组杆粒,为转染昆虫细胞(Sf9/Sf21)奠定了基础。
[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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