猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立与应用

发布时间：2018-10-05 08:55

【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。
[Abstract]:According to the published (PDCoV) N gene sequence of porcine Delta coronavirus (GenBank), specific primers and TaqMan probes were designed and synthesized to construct the standard plasmid. A real-time TaqMan quantitative PCR method for detecting PDCoV was established. The sensitivity, specificity and repeatability of the method were evaluated. The results showed that fluorescent signals were not detected using porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine Kub virus, porcine reproductive and respiratory syndrome virus and foot-and-mouth disease virus as templates. The coefficient of variation of circulating threshold Ct was lower than that of 2.66 脳 10 ~ 6 脳 10 ~ (5) and 2.66 脳 10~4copies/L plasmids. The results showed that the method had a good specificity and the coefficient of variation of circulating threshold Ct was lower than that of 2.66 脳 10 ~ (6) and 2.66 脳 10 ~ (6) 10~4copies/L. The slope of the standard curve was -3.461, and the correlation coefficient was 0.998. The results showed that there was a good linear relationship between the threshold value and the concentration of template, and the standard sample was diluted by gradient. The detection of plasmid DNA, with a minimum limit of 2.66 脳 10 ~ (-1) copies/L showed that the method had a good sensitivity. The results showed that the positive rate of PDCoV was 22.1, which was significantly higher than that of routine RT-PCR (11.9%), which indicated that this method could be applied to the clinical diagnosis and quantitative detection of PDCoV.
【作者单位】：甘肃农业大学生命科学技术学院;中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室;
【基金】：国家自然科学基金项目(31602095) 国家生猪产业体系项目(CARS-36-068) “十三五”国家重点研发计划(2016YFD0501505) 兰州兽医研究所统筹项目(Y2016CG23)
【分类号】：S852.651

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