基于催化发夹自组装技术的新型冠状病毒靶RNA快速荧光检测法的建立

Development of a Catalytic Hairpin Assembly-Based Fluorescent Assay for the Rapid Detection of SARS-CoV-2 Target RNA

目的建立一种基于催化发夹自组装技术(catalytic hairpin assembly,CHA)的新型冠状病毒(简称新冠病毒)靶RNA荧光检测法,实现对新冠病毒核酸的快速检测。方法根据CHA反应原理,选择新冠病毒核衣壳蛋白基因(N基因,NC_045512.2)上长24 nt的片段作为检测靶点(即靶RNA),设计合成发夹探针H1、H2,并在探针H1上修饰荧光基团和猝灭基团;在室温(25℃)下,向探针溶液中加入靶RNA引发CHA反应,通过检测反应过程中荧光强度的变化实现对靶RNA的快速检测;优化检测探针及反应条件,对方法的灵敏度和特异性进行评价。结果成功建立了针对新冠病毒靶RNA的荧光CHA法,可于室温下在30 min内完成检测,该法除具有优良的特异性、可准确区分靶RNA和发生单碱基突变的靶RNA之外,还具有良好的灵敏度,检出限为50 pmol/L。结论本研究所提出的方法实现了对新冠病毒靶RNA简单快速的检测,具有优良的特异性和灵敏度,有望通过进一步优化以用于临床新冠病毒核酸样本的快速检测。

Objective To develop a catalytic hairpin assembly(CHA)-based fluorescent assay for the detection of the target RNA of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),so as to realize the rapid nucleic acid testing of SARS-CoV-2.Methods A 24-nt segment of the SARS-CoV-2 nucleocapsid protein gene(N gene,NC_045512.2)was chosen as the target RNA and the hairpin motif 1(H1)and hairpin motif 2(H2)were designed based on the principle of CHA reaction.The H1 motif was labelled with a fluorophore group as well as a quencher group.When the target RNA was added to the hairpin motifs,CHA reaction was triggered at room temperature(25℃),which led to the amplification of fluorescence signal,thereby enabling the rapid detection of the target RNA.After the optimization of the hairpin motifs and the experimental conditions,the sensitivity and the specificity of the testing method were measured to evaluate its performance.Results We successfully constructed a CHA-based fluorescent assay specifically for the target RNA of SARS-CoV-2.With this method,testing could be completed at room temperature within 30 min.This testing method exhibited excellent specificity and could be used to accurately distinguish the perfectly-matched target RNA from the target RNA with single-base mutations.In addition,the testing method demonstrated good sensitivity,with a detection limit of 50 pmol/L.Conclusion The proposed assay enables the simple and rapid detection of the SARS-CoV-2 target RNA with excellent sensitivity and specificity,showing great promise for further optimization and subsequent clinical application for the rapid detection of SARS-CoV-2 nucleic acid.