【摘要】 根据Gen Bank登录的猪流行性腹泻病毒(PEDV)毒株(登录号KJ960180)的M基因保守序列,设计1对扩增片段大小为299 bp的特异性引物,经PCR扩增、克隆、测序鉴定后,提取质粒作为阳性标准品,建立了PEDV的SYBR GreenⅠ荧光定量RT-PCR检测方法。该方法在1.21×103~1.21×108拷贝/μL范围内呈现良好的线性,相关系数(R2)为0.999,扩增效率为99%,扩增产物的熔解曲线为单个特异峰,产物Tm值为85.5~86℃,最低检测限为1.21×101拷贝/μL。本研究建立的SYBR GreenⅠ实时荧光定量RT-PCR检测方法特异性强、重复性好、成本低且操作简单,适用于PEDV的早期诊断、定量研究和流行病学监测。更多还原

【Abstract】 According to the porcine epidemic diarrhea virus(PEDV)strain(Accession No. KJ960180) M conserved gene sequence available in Gen Bank,a pair of specific primers was designed and amplified 299 bp fragment.After PCR Amplification,cloning and sequencing,as a positive standard template to establish SYBR Green Ⅰ fluorescence RT-PCR detection for the quantization of PEDV.The standard curve generated had a wide dynamic range from 1.21×103~1.21×108 DNA copies/μL with a linear correlation(R2) of 0.999 and efficiency of 0.99.The melting curve analysis showed one specific peak with melting temprature(Tm) of 85.5 ~86 ℃,and no primer-dimers peak represented,and the sensitive degree is 1.21×101copies/μL,and showed excellent specificity and reproducibility.A SYBR Green Ⅰ fluorescent quantitative RT-PCR assay for detecting M gene of PEDV was developed for the early detection,quantitative analyzing and epidemiological surveillance.更多还原