Department of Pathogen Biology, Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, China.
Institute of Pathogenic Microbiology, Key Laboratories of Enteric Pathogenic Microbiology (Ministry of Health), Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
Sci Rep. 2017 Mar 21;7:44924. doi: 10.1038/srep44924.
Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A/H5, A/H7 and 2009A/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A/H5, A/H7, and 2009A/H1 influenza viruses, especially in resource-limited settings.
考虑到每年流感病毒感染造成的致命人类受害者和经济损失，迫切需要快速和现场检测流感病毒的方法。LAMP 是最常用的适合现场使用的核酸等温扩增技术。然而，对于多重 LAMP，目前仍然难以区分来自多个靶标的扩增子。在这里，我们开发了一种用于同时扩增三种主要流感病毒亚型（A/H5、A/H7 和 2009A/H1）的多重 RT-LAMP 检测方法。通过级联入侵反应和纳米颗粒杂交在单独的靶特异性检测管中进一步鉴定扩增子（称为 mRT-LAMP-IRNH）。该检测方法的分析灵敏度为所有三种 HA 亚型的 10 个拷贝的 RNA，特异性达到 100%。临床标本分析表明，该检测方法的综合敏感性和特异性分别为 98.1%和 100%。总的来说，mRT-LAMP-IRNH 检测方法可以作为一种节省成本的方法，利用简单的仪器来检测 A/H5、A/H7 和 2009A/H1 流感病毒，特别是在资源有限的环境中。
