Department of Life Science, Gachon University, 1342 Seongnam-Si, Gyeonggi-Do 13120, Korea.
Department of Microbiology, Institute for Viral Diseases, College of Medicine, Korea University, Seoul 02841, Korea.
Biosensors (Basel). 2021 Mar 19;11(3):88. doi: 10.3390/bios11030088.
A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 10 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.
一种快速且准确的现场病原体诊断测试，包括流感病毒，对于预防传染病的传播至关重要。两种类型的流感病毒，A 和 B 引起季节性流感流行，而 A 型则可引起流感大流行。为了专门检测甲型流感病毒（IAV）和乙型流感病毒（IBV），我们开发了一种基于成簇规律间隔短回文重复序列（CRISPR）和 CRISPR 相关（Cas）系统的检测方法。通过将逆转录重组酶聚合酶扩增（RT-RPA）和逆转录环介导等温扩增（RT-LAMP）相结合，CRISPR-Cas12a 内切酶靶向的 CRISPR 转报告子（DETECTR）检测到 IAV 和 IBV 的滴度低至每个反应 1 × 10 噬菌斑形成单位（PFU），且没有交叉反应。根据等温核酸扩增方法，检测 IAV 和 IBV 仅需 75 至 85 分钟，结果通过不需要额外分析设备的侧向流动条带检测进行验证。总之，我们的研究结果建立了基于 RT-RPA 和 RT-LAMP 的 DETECTR 诊断测试，用于使用荧光和侧向流动检测法快速、特异性和高灵敏度地检测 IAV 和 IBV。本研究开发的诊断测试可用于区分 IAV 和 IBV 感染，这是监测和预防流感流行和大流行传播所必需的。
