羊传染性脓疱实时荧光定量PCR方法的建立

发布时间：2018-01-29 06:02

  本文关键词： 羊传染性脓疱病毒 F1L基因 实时荧光定量PCR SYBR Green I TaqMan探针　出处：《内蒙古农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：羊传染性脓疱是由羊传染性脓疱病毒引起的山羊和绵羊的一种急性、接触性传染病。为了更快、更准确的诊断该病,本研究以羊传染性脓疱病毒F1L基因作为特异性基因,建立了两种羊传染性脓疱病毒实时荧光定量PCR检测方法,即：SYBR Green Ⅰ实时荧光定量PCR检测方法和TaqMan实时荧光定量PCR检测方法。根据GenBank已经发表的CEV参考毒株F1L基因序列,利用Primer Express 3.0软件针对两种方法设计合成两对特异性引物和一条TaqMan探针。分别构建两种方法的重组阳性质粒,作为标准品模板。优化两种方法的反应体系和反应参数,分别绘制标准曲线,评价标准品的相关性和扩增效率,检验所建立的两种实时荧光定量PCR反应体系的重复性、特异性和敏感性。对11例羊传染性脓疱阳性病例病料进行了检测,验证所建立的两种方法的实用性。结果显示：SYBR GreenⅠ实时荧光定量PCR检测方法标准曲线的相关系数R2=0.99,扩增效率E=1.18,最低检出下限模板浓度为65.47copies/μL,组内和组间变异系数分别为0.17%～0.59%和0.74%-1.25%；TaqMan实时荧光定量PCR检测方法标准曲线的相关系数R2=0.997,扩增效率E=1.08,最低检出下限模板浓度为13.40copies/μL,组内和组间变异系数分别为0.28%-0.55%和0.45%-1.31%。两种检测方法重复性好,特异性强,敏感性高,均与山羊痘活疫苗、口蹄疫O型、亚洲Ⅰ型二价灭活疫苗及小反刍兽疫病毒疫苗不发生交叉反应；对收集自不同地区的11例羊传染性脓疱病料的检测结果均为阳性。
[Abstract]:Infectious pustules in sheep are an acute, contagious infectious disease in goats and sheep caused by the infectious pustular virus in sheep. In order to diagnose the disease more quickly and accurately. In this study, the F1L gene of sheep infectious pustular virus was used as the specific gene, and two real-time fluorescent quantitative PCR methods were established for the detection of sheep infectious pustular virus. That is: SYBR Green. I Real-time fluorescence quantitative PCR detection method and TaqMan real-time fluorescence quantitative PCR detection method. According to the F1L gene sequence of CEV reference strain published by GenBank. Two pairs of specific primers and one TaqMan probe were designed and synthesized by using Primer Express 3.0 software for the two methods. The recombinant positive plasmids of the two methods were constructed respectively. As standard sample template, optimize the reaction system and parameters of the two methods, draw the standard curve, evaluate the correlation and amplification efficiency of the standard product. The reproducibility, specificity and sensitivity of two real-time fluorescent quantitative PCR reaction systems were tested in 11 sheep infectious pustular positive cases. The results showed that the correlation coefficient of the standard curve of the real-time fluorescence quantitative PCR method for the detection of 1: SYBR Green 鈪，

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