鉴别GSFV与BVDV1双重实时荧光定量PCR方法的建立
发布时间：2018-08-11 13:23
【摘要】：本实验为了建立一种快速准确、特异性强、敏感性高的鉴别CSFV和BVDV的双重实时荧光PCR方法,根据GenBank上已发表的猪瘟病毒和牛病毒性腹泻-黏膜病1型病毒的全基因序列,进行对比分析,分别设计合成两对能特异性扩增CSFV和BVDV1的2对引物及2条探针。制备标准阳性质粒DNA模板,对双重实时荧光定量PCR反应参数和反应条件进行优化,并制备实时荧光PCR标准曲线,评价所建立的双重实时荧光定量PCR反应体系的敏感性、特异性、重复性,并用本试验建立的鉴别CSFV和BVDV1的双重实时荧光定量PCR方法对86份临床样品进行检测。结果显示：引物浓度400 nmol/L,探针浓度为300 nmol/L,退火温度58℃C时所建立的双重实时荧光定量PCR方法达到最佳的反应效果。并且此诊断方法可以特异的检测CSFV和BVDV1,并能与绵羊痘病毒、山羊痘病毒、羊口疮病毒、小反刍兽疫病毒、牛传染性鼻气管炎病毒区分。另外,此建立的方法Ct值的变异系数均小于0.01,CSFV和BVDV1的最小检出量分别为1.04x102拷贝数／μL、1.15×102拷贝数/μL。表明所建立的双重实时荧光PCR方法具有较强的稳定性、较高的敏感性。运用所建立的PCR方法检测临床样品,可以快速、准确的检测CSFV和BVDV1。
[Abstract]:In order to establish a rapid, accurate, specific and sensitive dual real-time fluorescent PCR method for the identification of CSFV and BVDV, the whole gene sequence of CSFV and BVD-1 virus published on GenBank was established. Two pairs of primers and two probes for specific amplification of CSFV and BVDV1 were designed and synthesized. The standard positive plasmid DNA template was prepared to optimize the reaction parameters and reaction conditions of double real-time fluorescent quantitative PCR, and the standard curve of real-time fluorescent PCR was prepared to evaluate the sensitivity and specificity of the dual real-time fluorescent quantitative PCR reaction system. The reproducibility of 86 clinical samples was detected by double real time fluorescence quantitative PCR method which was established in this study to identify CSFV and BVDV1. The results showed that the double real-time PCR method with primer concentration of 400 nmol / L, probe concentration of 300 nmol / L and annealing temperature of 58 鈩，
本文编号：2177118
[Abstract]:In order to establish a rapid, accurate, specific and sensitive dual real-time fluorescent PCR method for the identification of CSFV and BVDV, the whole gene sequence of CSFV and BVD-1 virus published on GenBank was established. Two pairs of primers and two probes for specific amplification of CSFV and BVDV1 were designed and synthesized. The standard positive plasmid DNA template was prepared to optimize the reaction parameters and reaction conditions of double real-time fluorescent quantitative PCR, and the standard curve of real-time fluorescent PCR was prepared to evaluate the sensitivity and specificity of the dual real-time fluorescent quantitative PCR reaction system. The reproducibility of 86 clinical samples was detected by double real time fluorescence quantitative PCR method which was established in this study to identify CSFV and BVDV1. The results showed that the double real-time PCR method with primer concentration of 400 nmol / L, probe concentration of 300 nmol / L and annealing temperature of 58 鈩，
本文编号：2177118
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