基因 VI 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，舒波 ^1，2，于晓慧 ¹，克军宏 ^1，3，邢安琪 ^1，4，彭真奇 ^1，5，刘华雷 ¹

Abstract

基因 Ⅵ 型新城疫病毒（NDV）是目前造成我国鸽新城疫流行的主要基因型。国家新城疫参考实验室监测数据显示，近年来鸽源 NDV 分离率呈上升趋势。为实现基因 Ⅵ 型 NDV 快速检测与精确定量，本研究针对我国流行基因 Ⅵ 型 NDV 的 F 基因保守区设计特异性引物与探针，建立反转录数字 PCR（RT-dPCR）检测方法，并系统评价该方法的灵敏度、特异性及重复性。结果表明，所建立的 RT-dPCR 方法线性关系良好、灵敏度优异，最低检测限为1.97 copies/μL；特异性良好，与其他基因型新城疫病毒强毒株及常见禽类病原病毒均无交叉反应；重复性可靠，变异系数为2.2 %。采用该方法对临床收集的180份鸽口咽 / 泄殖腔拭子样品进行检测，检测结果与病毒分离结果吻合。综上，本研究建立的 RT-dPCR 方法灵敏度高、特异性强、重复性佳，适用于基因 Ⅵ 型 NDV 的快速筛查与精准定量，也可为基因 Ⅵ 型 NDV 核酸标准物质的研制提供技术支撑。

Newcastle disease virus (NDV) genotype Ⅵ is the predominant genotype responsible for the prevalence of pigeon Newcastle disease (ND) in China. Surveillance data from the National Newcastle Disease Reference Laboratory revealed that the isolation rate of pigeon-origin NDV has increased in recent years. To achieve rapid detection and accurate quantification of NDV genotype Ⅵ, specific primers and probes were designed targeting the conserved region of the F gene of prevalent NDV genotype Ⅵ strains, and a reverse transcription digital PCR (RT-dPCR) assay was established. Subsequently, its sensitivity, specificity and repeatability were systematically evaluated. The results showed that this RT-dPCR assay exhibited favorable linearity and high sensitivity, with a limit of detection of 1.97 copies/μL. It had strong specificity with no cross-reactivity against virulent strains of other NDV genotypes and common avian pathogenic viruses, and presented good repeatability with a coefficient of variation of 2.2 %. A total of 180 clinical pigeon oropharyngeal and cloacal swab samples were tested, and the detection results were consistent with those obtained by virus isolation. In summary, the established RT-dPCR assay possesses high sensitivity, specificity and repeatability, which is suitable for rapid detection and precise quantification of NDV genotype Ⅵ, and also provides a basis for the development of nucleic acid reference materials for NDV genotype Ⅵ.