【摘要】 根据GenBank中已公布的猪德尔塔冠状病毒(PDCoV)N基因序列设计并合成特异的引物和TaqMan探针,构建质粒标准品,以标准品为模板建立了检测PDCoV的TaqMan实时荧光定量PCR方法,并对该方法的敏感性、特异性及重复性进行了评价。结果显示,以猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪库布病毒、猪繁殖与呼吸综合征病毒和口蹄疫病毒为模板时均未检测到荧光信号,表明该方法具有良好的特异性;用2.66×10~6、2.66×10~5和2.66×10~4copies/L这3个不同浓度的质粒标准品进重复试验,循环阈值Ct的变异系数均低于2%,表明该方法具有良好的特异性;标准曲线斜率为-3.461,相关系数为R~2=0.998,表明阈值和模板浓度之间具有良好的线性关系。10倍梯度稀释质粒标准品,检测的最低限度为2.66×10~1 copies/L的质粒DNA,表明此方法具有良好的敏感性。利用该方法对194份临床猪粪便样品进行了检测,结果显示PDCoV阳性率为22.1%,明显高于常规RT-PCR的11.9%,表明该方法可应用于PDCoV的临床诊断及定量检测。更多还原

【Abstract】 The specific primer and TaqMan probe for PDCoV were designed and synthesized according to reference sequences published on GenBank,and plasmid standards were constructed.The TaqMan real time fluorescent PCR for porcine deltacoronavirus was established using standard samples and the parameters of assay were optimized including sensibility,specificity and repeatability.The results showed that no signals were detected when using PEDV,TGEV,PKV,PRRSV and FMDV as templates and indicated the assay had good specificity.The variation coefficient of cycle threshold Ctwas less than 2% in repeated experiments using 2.66×10~6,2.66×10~5 and 2.66×10~4 copies/L of standard samples as templates,which indicated the assay had good repeatability.The standard curve slope and correlation coefficient (R~2) were-3.461 and 0.998,respectively,which indicated that there existed good linear relationship between threshold value and template concentration.Ten-fold serial standard sample dilutions were detected using this assay and the results showed that the detection limit was 2.66×10~1 copies/L plasmid DNA and indicated this assay had higher sensitivity.194 clinical swine feces were detected using this assay and the results showed the positive rate of PDCoV was 22.1%,significantly higher than that by conventional RT-PCR (11.9%).In conclusion,this assay can be applied to clinical diagnosis and quantitative analysis for PDCoV.更多还原