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Abstract：
The COVID-19 pandemic has unfortunately demonstrated how easily infectious diseases can spread and harm human life and society. As of writing, pandemic has now been on-going for more than one year. There is an urgent need for new nucleic acid-based methods that can be used to diagnose pathogens early, quickly, and accurately to effectively impede the spread of infections and gain control of epidemics. We developed a flap probe-based isothermal nucleic acid amplification method that is triggered by recombinant FEN1-Bst DNA polymerase, which-through enzymatic engineering-has both DNA synthesis, strand displacement and cleavage functions. This novel method offers a simpler and more specific probe-primer pair than those of other isothermal amplifications. We tested the method\'s ability to detect SARS-CoV-2 (both ORF1ab and N genes), rotavirus, and Chlamydia trachomatis. The limits of detection were 10 copies/μL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene, and 100 copies/μL for SARS-CoV-2 ORF1ab gene. There were no cross-reactions among 11 other common pathogens with characteristics similar to those of the test target, and the method showed 100% sensitivity and 100% specificity in clinical comparisons with RT-PCR testing. In addition to real-time detection, the endpoint could be displayed under a transilluminator, which is a convenient reporting method for point-of-care test settings. Therefore, this novel nucleic acid senor has great potential for use in clinical diagnostics, epidemic prevention, and epidemic control.
摘要：
不幸的是，COVID-19大流行证明了传染病是多么容易传播和危害人类生命和社会。作为写作,大流行已经持续了一年多。迫切需要可用于早期诊断病原体的新的基于核酸的方法，快,并准确地有效阻止感染的传播并控制流行病。我们开发了一种基于瓣探针的等温核酸扩增方法，该方法由重组FEN1-BstDNA聚合酶触发，通过酶工程，既有DNA合成，链置换和裂解功能。与其他等温扩增的方法相比，这种新方法提供了更简单，更特异的探针-引物对。我们测试了该方法检测SARS-CoV-2（ORF1ab和N基因）的能力，轮状病毒，和沙眼衣原体.轮状病毒的检测限为10拷贝/μL，C.沙眼，和SARS-CoV-2N基因，和100拷贝/μL的SARS-CoV-2ORF1ab基因。其他11种常见病原体之间没有交叉反应，其特征与测试目标相似，在与RT-PCR检测的临床比较中，该方法显示出100％的灵敏度和100％的特异性。除了实时检测，端点可以显示在过渡器下面，这是即时测试设置的便捷报告方法。因此,这种新型核酸传感器在临床诊断中具有巨大的应用潜力，防疫,和流行病控制。
