摘要
基因Ⅵ型新城疫病毒(NDV)是导致我国当前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表明,鸽NDV分离率近年来有上升趋势。为了实现基因Ⅵ型NDV快速检测和精确定量,针对国内流行的基因Ⅵ型NDV F基因保守区域设计特异性引物和探针,建立了反转录数字PCR(RT-dPCR)方法,并对该方法的灵敏度、特异性和重复性进行了评估。结果显示,建立的RT-dPCR方法线性关系良好,灵敏度高,最低检测限为1.97 copies/μL;特异性强,与其他基因型NDV强毒株和常见禽病病毒无交叉反应;重复性好,变异系数为2.2%。利用本方法对临床采集的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一致。以上结果表明,本研究建立的RT-dPCR方法灵敏度高、特异性强、重复性好,可用于基因Ⅵ型NDV的快速检测和精准定量,同时也为基因Ⅵ型NDV核酸标准物质研制奠定了基础。
Newcastle disease virus(NDV)genotype Ⅵ was the major genotype that cause the prevalence of pigeon Newcastle disease(ND)in China,and the isolation rate of NDV in pigeons has been increasing in recent years as indicated by the surveillance data collected by National Newcastle Disease Reference Laboratory. In order to rapidly detect and accurately quantify NDV genotype Ⅵ,specific primers and probes were designed based on the conserved region of NDV genotype Ⅵ F gene,then a reverse transcription digital PCR(RT-dPCR)assay was established,followed by evaluation on its specificity,sensitivity and repeatability. The results showed that the established method was with good linear relation,high sensitivity,and a minimum detection limit of 1.97 copies/μL,as well as strong specificity as no cross reaction with other genotype NDV virulent strains or common avian disease viruses was observed,and good repeatability with the variable coefficient of 2.2%. 180 pigeon oropharyngeal/cloacal swab samples collected clinically were detected by the established method,and the results were consistent with those of virus isolation. In conclusion,the established RT-dPCR was with high specificity,sensitivity and repeatability,which could be used for rapid detection and accurate quantification of NDV genotype Ⅵ,and also lay a foundation for future development of reference materials of NDV genotype Ⅵ nucleic acid.
作者
王静静
舒波
于晓慧
克军宏
邢安琪
彭真奇
刘华雷
Wang Jingjing;Shu Bo;Yu Xiaohui;Ke Junhong;Xing Anqi;Peng Zhenqi;Liu Hualei(China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China;Jiangxi Agricultural University,Nanchang,Jiangxi 330045,China;Tarim University,Alaer,Xinjiang 843300,China;Ningxia University,Yinchuan,Ningxia 750021,China;Anhui Agricultural University,Hefei,Anhui 230036,China)
出处
《中国动物检疫》
CAS
2022年第9期115-120,共6页
China Animal Health Inspection
基金
国家重点研发计划项目(2016YFD0500800)
中国动物卫生与流行病学中心创新基金项目。