College of Animal Science and Veterinary Medicine, Shandong Agricultural University, #61 Dai Zong Avenue, Tai'an, Shandong 271018, China.
Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
Biosens Bioelectron. 2016 Dec 15;86:255-261. doi: 10.1016/j.bios.2016.06.063. Epub 2016 Jun 22.
Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.
禽流感病毒（AIV）亚型 H5N1 引起了特别关注，因为它是动物和公共卫生系统的持续威胁。AIV H5N1 感染引起的病毒血症可能会增加人与人之间血源性传播的风险。因此，需要快速评估和实施 AIV H5N1 病毒血症的筛选措施，以便对这一潜在的大流行威胁做出快速反应。本报告介绍了一种基于免疫测定的逆转录环介导等温扩增（immuno-RT-LAMP）检测方法，用于快速检测全血样本中的 AIV H5N1。使用涂有 H5 亚型单克隆抗体的 PCR 管，从血液样本中特异性捕获 AIV H5N1 病毒粒子。在热裂解步骤后，使用 RT-LAMP 在实时 PCR 仪器上进行定量分析，或在水浴系统中进行终点分析，对释放的病毒 N1 基因进行指数扩增。新开发的 immuno-RT-LAMP 检测方法的检测限低至 1.62×10(1) 50%胚胎感染剂量/mL 的病毒，无论是常规样本还是模拟病毒血症样本。与非 H5N1 流感病毒或其他禽病毒均无交叉反应。使用内和间试验进行了该测定的重现性确认，其变异性范围为 1.05%至 3.37%。我们的结果表明，immuno-RT-LAMP 是一种新颖、有效的即时护理病毒识别解决方案，可用于快速诊断和监测血液样本中的 AIV H5N1。
