鸡传染性法氏囊病病毒SYBR GreenⅠ实时荧光定量RT-PCR检测方法的建立

Detection of Infectious Bursal Disease Virus Using RT-PCR SYBR GreenⅠ Technology

摘要: RT-PCR扩增IBDV的VP5基因保守片段,将其克隆到pMD18-T载体,经测序鉴定得到阳性质粒。以阳性质粒为标准品,建立SYBR GreenⅠ实时荧光定量RT-PCR的标准曲线和溶解曲线。结果表明,IBDV荧光定量PCR的标准曲线Ct值与3.2910～3.29108之间的病毒基因拷贝数呈现良好的线性关系。该方法灵敏度可达33拷贝,且特异性和重复性好。SYBR GreenⅠ实时荧光定量RT-PCR方法可用于IBDV的病原诊断和病毒的定量分析。

Abstract: Conservative fragment of VP5 gene was amplified by RT-PCR and cloned into pMD18-T vector.After sequencing,the positive recombinant plasmid was acquired and used to establish standard and melt curves of the real-time fluorescent quantitative RT-PCR.The standard curve for the threshold cycle and viral genomic copy number ranging from 3.2910～3.29108 were linear.The sensitivity of detection was 33 copies.It was concluded that the real time RT-PCR SYBR GreenⅠ technology was highly specific and repeatable for IBDV diagnostics and quantification.