基因 Ⅶ.2 亚型新城疫病毒反转录数字 PCR 检测方法的建立与应用

王静静 ¹，克军宏 ²，于晓慧 ¹，彭真奇 ³，邢安琪 ⁴，刘华雷 ¹

Abstract

基因 Ⅶ 型新城疫病毒（NDV）是严重危害我国家禽养殖业健康发展的主要病原之一。国家新城疫参考实验室监测表明，目前国内 NDV 流行毒株已由基因 Ⅶ.1.1 亚型转变为 Ⅶ.2 亚型。为实现基因 Ⅶ.2 亚型 NDV 的快速检测和精准定量，针对其国内流行毒株F基因保守区域设计了特异性引物和探针，建立了一种可快速检测基因 Ⅶ.2 亚型 NDV 的反转录数字 PCR（RT-dPCR）方法，并评估了该方法的敏感性、特异性和重复性。结果显示：建立的 RT-dPCR 方法检测下限为1.30 copies/μL，敏感性较高；特异性强，与基因 Ⅵ 型、Ⅻ 型 NDV 以及其他常见禽病病毒（禽流感病毒、鸡传染性支气管炎病毒、传染性喉气管炎病毒和禽腺病毒）无交叉反应；重复性试验变异系数为2.6 %，小于3 %，重复性好。利用本方法对从活禽市场采集的 200 份鸡口咽 / 泄殖腔拭子样品进行检测，发现检测结果与病毒分离鉴定结果一致。结果表明，本研究建立的 RT-dPCR 方法具有良好的敏感性、特异性、重复性和准确性，可用于基因 Ⅶ.2 亚型 NDV 的快速检测和精准定量，同时也为其核酸标准物质的研制奠定了基础。

Genotype Ⅶ Newcastle disease virus (NDV) is one of major pathogens seriously endangering poultry industry in China. Monitoring conducted by National Newcastle Disease Reference Laboratory revealed that current NDV strains have evolved from subtype Ⅶ.1.1 to Ⅶ.2. In order to rapidly detect and accurately quantify Ⅶ.2 subtype NDV, based on the conserved region of F gene of its domestic strains, specific primers and probes were designed to establish a reverse transcription digital PCR (RT-dPCR), followed by evaluation on its sensitivity, specificity and repeatability. The results showed that the established method, with high sensitivity, could detect at a low limit of 1.30 copies/μL; with strong specificity, failed to crossly react with genotypes Ⅵ, Ⅶ and NDV and other common avian disease viruses (including avian influenza virus, avian infectious bronchitis virus, infectious laryngotracheitis virus and avian adenovirus); and with good repeatability, the coefficient of variation (CV) of repeatability test was 2.6 %, less than 3 %. 200 oropharyngeal/cloacal swab samples collected from live poultry markets were tested by the method, from which, the results were consistent with those of virus isolation and identification. In conclusion, the method was with high specificity, sensitivity and repeatability, and could be used for rapid detection and accurate quantification of sub-genotype VII.2 NDVs, and would lay a foundation for future development of related reference materials.