Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.
Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.
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开发重组酶聚合酶扩增结合侧流试纸法检测牛短暂热病毒
DOI：
10.1016/j.mcp.2017.12.003
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发表时间：
2018-04
影响因子：
3.3
通讯作者：
He H
中科院分区：
文献类型：
--
作者：
Hou P;Zhao G;Wang H;He C;Huan Y;He H
牛流行性出血热病毒(BEFV)是引起牛流行性出血热(BEF)的病原体，其流行/暴发的数量不断增加，对畜牧业造成了严重的危害。因此，建立BEFV的快速检测方法势在必行。在这项研究中，我们描述了侧流试纸等温重组酶聚合酶扩增(LFD-RPA)检测BEFV的方法的发展。针对特定的G基因设计了RPA引物和LF探针，用肉眼可以在简单的侧流试纸上观察到扩增产物。扩增反应在38 °C，20 分钟，LFD孵育时间5 分钟内进行。该方法的检出限为8拷贝/次，与牛病毒性腹泻病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞病毒、牛冠状病毒、牛副流感病毒3型、牛水泡性口炎病毒等牛传染性疾病病毒无交叉反应。此外，对128例临床标本进行了检测，并与常规RT-PCR、实时定量(Q)PCR检测结果进行了比较。结果表明，BEFV LFD-RPA与实时荧光定量聚合酶链式反应的符合率为96.09%(123/128)，高于常规RT-PCR。RPA与LFD联合检测可能为BEFV感染暴发的诊断提供了一种快速、敏感的替代方法。首次建立了RPA-LFD联合检测BEFV的方法。检测过程在30 分钟内完成，肉眼可见。RPA与LFD联合检测可能为BEFV的诊断提供了一种新的选择。
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. RPA combined with LFD assay was developed first time to detect BEFV. The detection from cDNA could be completed within 30 min and be easily visualized with the naked eyes. The RPA combined with LFD assay probably provides a alternative for diagnosis of BEFV.
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牛短暂热病毒新遗传机制、起源和种群动态
影响因子：
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