The novel coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world, which has exposed humanity to unprecedented economic, social and health impacts. To achieve efficient and accurate detection of SARS-CoV-2 on site, we developed and verified a rapid and sensitive fluorescence lateral flow immunoassay based on the innovative enhanced strand exchange amplification (ESEA-LFIA) in this study. With good amplification efficiency for short-sequence targets, ESEA is an ideal choice for the point-of-care testing of SARS-CoV-2 with a high mutation rate. ESEA reaction can be completed in one step and verified by restriction enzyme digestion. The design consisting of three working primers greatly improved the amplification efficiency. Amplification of the target sequences of the RdRP and N genes can be accomplished under the same reaction conditions, and does not require expensive instruments. The sensitivity of the ESEA-LFIA assay targeting the RdRP and N genes was 90 copies per μL and 70 copies per μL, respectively. Specificity tests showed that the novel assay can specifically detect SARS-CoV-2, and had no cross-reactivity with 9 closely-related human pathogenic coronaviruses and other common respiratory pathogens with similar clinical manifestations. The cutoff values of the RdRP and N gene assays are 11 and 12, respectively, and the assays can be completed within 1 h. The novel strategy proposed in this study is a sensitive and specific method for the rapid detection of SARS-CoV-2, and is suitable as an effective potential bioanalytical tool to respond to future regional or global outbreaks of emerging infectious pathogens with high mutation rates.

中文翻译：

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三引物增强链交换扩增结合快速侧流荧光免疫分析检测SARS-CoV-2

由严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）引起的 2019 年新型冠状病毒病在全球迅速蔓延，使人类面临前所未有的经济、社会和健康影响。为了在现场实现对 SARS-CoV-2 的高效准确检测，我们在本研究中开发并验证了一种基于创新增强链交换扩增 (ESEA-LFIA) 的快速灵敏的荧光侧流免疫测定法。ESEA 对短序列靶标具有良好的扩增效率，是对具有高突变率的 SARS-CoV-2 进行即时检测的理想选择。ESEA反应可以一步完成，并通过限制性内切酶消化进行验证。由三个工作引物组成的设计大大提高了扩增效率。RdRP和N基因可以在相同的反应条件下完成，不需要昂贵的仪器。ESEA-LFIA 检测针对RdRP和N基因的灵敏度分别为每微升 90 拷贝和每微升 70 拷贝。特异性测试表明，该新方法可特异性检测SARS-CoV-2，与9种密切相关的人类致病冠状病毒和其他临床表现相似的常见呼吸道病原体无交叉反应。RdRP和N的截止值基因检测分别为11次和12次，可在1小时内完成检测。本研究提出的新策略是一种快速检测 SARS-CoV-2 的灵敏而特异的方法，适合作为一种有效的潜在生物分析工具，以应对未来区域或全球爆发的具有高突变率的新兴传染性病原体。