新型鹅呼肠孤病毒TaqMan荧光定量RT-PCR检测方法的建立

Development of A TaqMan Real-Time RT-PCR Assay for Detection of Novel Goose Reovirus

2020年以来,在我国养鹅地区出现了一种由新型鹅呼肠孤病毒(Novel goose reovirus,NGRV)引起的以肝脾点状白色灶性坏死为主要病变特征的鹅传染性疾病。为了快速有效地检测NGRV,本试验针对禽呼肠孤病毒的λC基因设计引物和探针,建立了基于TaqMan探针荧光定量PCR方法,结果显示:该方法特异性强,对鹅细小病毒、坦布苏病毒、鹅圆环病毒、番鸭呼肠孤病毒、新型鸭呼肠孤病毒和鹅星状病毒等病原不存在交叉反应;该方法具有较高的敏感性,最低可检测限为56.6拷贝/μL,比常规PCR方法敏感10倍;该方法的重复性良好,组内和组间变异系数小于2%。对37份疑似新型鹅呼肠孤病毒病的临床样品检测结果显示,荧光定量RT-PCR和RT-PCR检测出阳性样品均为13份,两者符合率为100%。测序结果表明,13份阳性样品均为NGRV。本试验成功建立了NGRV TaqMan荧光定量RT-PCR检测方法,为新型鹅呼肠孤病毒病的流行病学调查和防控奠定了坚实的基础。

Since July 2020,an infectious disease characterized by white focal necrosis in livers and spleens has spreading widely on goose farms in many regions of China.In this study,a TaqMan real-time RT-PCR method was developed using specifi c primers and probes according to theλC gene of Novel goose reovirus(NGRV).The result showed that the TaqMan real-time RT-PCR method had no cross-reaction with many pathogens such as GPV,TMUV,GoCV,MDRV,NDRV and GAstV.The method had a higher sensitivity and the detection limit was 56.6 copies/μL.The intra-and inter-assay coeffi cients of variation were less than 2%.Total 37 clinical samples were tested using this TaqMan real-time RT-PCR method and a conventional RT-PCR.As a result,13 positive samples were detected by both methods and the consistent rate of two assays was 100%.The TaqMan real-time RT-PCR method developed in this study laid a solid foundation for the epidemiological investigation and prevention and control of novel goose reovirus disease.