Experimental Animal Research Center, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
J Virol Methods. 2013 Feb;187(2):274-7. doi: 10.1016/j.jviromet.2012.11.020. Epub 2012 Nov 23.
Rabbit hemorrhagic disease virus (RHDV) causes haemagglutination and severe liver damage, with a high mortality rate. To develop a rapid and sensitive method for the surveillance of RHDV, a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of four primers specific for the VP60 gene segment of RHDV. The established assay was performed at 64°C for 40 min under isothermal conditions, and the results were visualized directly by electrophoresis or as fluorescent signals under ultraviolet light. The detection limit of the RT-LAMP assay was 10 copies of viral RNA per reaction, which was comparable to quantitative real-time RT-PCR, and 100-fold more sensitive than standard RT-PCR. Furthermore, seven viral RNAs of field isolates in China could be detected successfully using this assay. Overall, the newly established RT-LAMP assay indicates the potential usefulness of the technique as a simple, rapid and sensitive procedure, and can visually detect RHDV infection without the need for any specialized equipment.
兔出血症病毒（RHDV）可引起红细胞凝集和严重肝损伤，死亡率较高。为了开发一种快速灵敏的兔出血症病毒监测方法，本研究使用针对 RHDV VP60 基因片段的 4 条特异性引物建立了一步法逆转录环介导等温扩增（RT-LAMP）检测方法。该检测方法在 64°C 下等温反应 40min，结果可直接通过电泳或在紫外光下观察荧光信号进行可视化判断。RT-LAMP 检测方法的检测限为每个反应 10 个拷贝的病毒 RNA，与实时定量 RT-PCR 相当，但比标准 RT-PCR 灵敏 100 倍。此外，该方法还可成功检测中国 7 株田间分离株的病毒 RNA。总之，新建立的 RT-LAMP 检测方法具有简单、快速和灵敏的特点，可用于直观检测兔出血症病毒感染，无需任何特殊设备。
