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通过比对新城疫病毒强、弱毒株F基因序列,根据其在F0裂解位点的差异以及F基因保守序列设计合成NDV-V-probe和NDV-L-probe探针组和NDV-F、NDV-R引物组,以基因Ⅶ型NA-1株和基因Ⅱ型LaSota疫苗株病毒RNA作为定量检测的标准品,建立检测方法。对所建立的标准曲线进行分析,相关系数R2均为0.997,线性关系良好。通过计算变异系数CV/%分别为0.034%和0.027%,均小于1%,说明稳定性良好。对禽类常见病毒IBV、H9亚型AIV检测未发现有交叉反应,特异性好、重复性佳。结果表明:成功建立了新城疫病毒强、弱毒双重荧光定量RT-PCR的检测方法,实现了从分子水平上快速鉴别强、弱毒力的新城疫病毒,也可快速区分野毒感染动物和疫苗接种动物,减少不必要的经济损失。
NDV-V-probe and NDV-L-probe probes were designed and synthesized according to the F gene sequence of strong and weak virulent and virulent strains of Newcastle disease virus according to their differences in F0 cleavage sites and the conserved sequence of F gene. NDV-F, NDV -R primer set, the detection method was established by using the genotype Ⅶ NA-1 strain and the genotype II LaSota vaccine virus as the standard of quantitative detection. The established standard curve analysis, the correlation coefficient R2 are 0.997, a good linear relationship. By calculating the coefficient of variation CV /% were 0.034% and 0.027%, were less than 1%, indicating good stability. Common avian viruses IBV, H9 subtype AIV test found no cross-reaction, good specificity, good repeatability. The results showed that the detection of Newcastle disease virus (NDV) strong and attenuated double fluorescent quantitative RT-PCR was successfully established, and the rapid and differential virulence of Newcastle disease virus at the molecular level was achieved. It was also possible to quickly distinguish the wild-type infected animals from the vaccinated Animals, reduce unnecessary economic losses.