摘要目的:基于逆转录重组酶介导的恒温扩增（reverse transcriptional recombinase aided amplification，RT-RAA）技术，建立一种快速，灵敏，简便的SARS-CoV-2棘突蛋白（S）H146Y突变检测方法。方法:针对SARS-CoV-2棘突蛋白H146Y突变区域设计RT-RAA特异性引物和荧光探针，使用优化的RT-RAA方法进行恒温扩增检测，评价方法的灵敏度和特异性，并与二代测序技术进行比较。结果:基于荧光信号的RT-RAA方法能在39 ℃，30 min完成检测，SARS-CoV-2 S蛋白H146（S-H146）野生株和Y146（S-Y146）突变株检测限均为10 copies/μL，能够依据荧光值明显区分S-H146毒株与S-Y146毒株的重组质粒或临床样本，且和五种常见呼吸道感染病毒无交叉反应，与二代测序技术方法一致性高。结论:建立的RT-RAA荧光检测方法具有灵敏度高，特异性强，适用于SARS-CoV-2 S蛋白H146Y突变株的快速检测，为基层医疗机构提供了新的鉴定方法。

abstractsObjective:To develop a rapid and specific method to detect H146Y mutation in the severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein based on reverse transcriptional recombinase aided amplification (RT-RAA) method.Methods:The specific RT-RAA primers and fluorescence probe were designed for the mutation segment of H146Y mutation in SARS-CoV-2 S protein. The optimized RT-RAA method was used for amplification and detection and to evaluate the sensitivity and the specificity of the method. The results were compared with the next generation sequencing method.Results:The RT-RAA method based on fluorescence detection completed the detection within 30 minutes at 39℃. For 146 amino acid residue in SARS-COV-2 S protein, the detection limits for both wild type (S-H146) and mutant (S-Y146) were 10 copies/μL. The strains containing H146 and Y146 could be distinguished in the recombinant plasmid or clinical samples significantly based on fluorescence values. There was no cross-reaction with other five respiratory tract infectious viruses. The RT-RAA fluorescence assay showed high consistency with the next generation sequencing method.Conclusions:The developed RT-RAA fluorescence assay showed high sensitivity and specificity for quickly detecting H146Y mutation in SARS-CoV-2 S protein, and provided a new detection method for community medical institutions.