Key Laboratory of Animal Resistant Biology of Shandong, Ruminant Diseases Research Center, College of Life Sciences, Shandong Normal University, Jinan, China.
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, China.
Mol Cell Probes. 2018 Apr;38:31-37. doi: 10.1016/j.mcp.2017.12.003. Epub 2017 Dec 26.
Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.
牛暂时热病毒（BEFV），被鉴定为牛暂时热（BEF）的病原体，导致越来越多的疫情爆发，并对畜牧业造成重大危害。因此，快速检测方法对于 BEFV 的诊断至关重要。本研究描述了横向流动条带式等温重组聚合酶扩增（LFD-RPA）检测 BEFV 的方法的建立。RPA 引物和 LF 探针是针对特定的 G 基因设计的，扩增产物可以用肉眼在简单的横向流动条带上可视化。扩增反应在 38°C 下进行 20 分钟，LFD 孵育时间为 5 分钟。该检测方法的检测限为每个反应 8 个拷贝，与其他牛传染性病毒如牛病毒性腹泻病毒、传染性牛鼻气管炎病毒、牛呼吸道合胞体病毒、牛冠状病毒、牛副流感病毒 3 型、牛水疱性口炎病毒无交叉反应。此外，该方法用 128 份临床标本进行了检测，并将检测结果与常规 RT-PCR、实时定量(q)PCR 进行了比较。结果表明，BEFV LFD-RPA 与实时 qPCR 的符合率为 96.09%（123/128），高于常规 RT-PCR。RPA 与 LFD 联合检测可能为 BEFV 感染爆发的快速灵敏诊断提供了一种替代方法。
