猪流行性腹泻病毒实时荧光定量RT-PCR检测方法的建立与应用

Development and Application of Real-time Quantitative RT-PCR Technique for Detecting Porcine Epidemic Diarrhea Virus

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1.21×10^3~1.21×10^8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1.21×10^1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。

According to the porcine epidemic diarrhea virus （PEDV）strain （Accession No. KJ960180） M conserved gene sequence available in GenBank,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 GreenⅠfluorescence RT-PCR detection for the quantization of PEDV.The standard curve generated had a wide dynamic range from 1.21×10^3~1.21×10^8 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 1.21×10^1 copies/μL,and showed excellent specificity and reproducibility.A SYBR GreenⅠfluorescent quantitative RT-PCR assay for detecting M gene of PEDV was developed for the early detection,quantitative analyzing and epidemiological surveillance.