SARS-CoV-2棘突蛋白H146Y突变的逆转录重组酶介导的等温扩增方法建立  

Establishment of a RT-RAA method for H146Y mutation in SARS-CoV-2 spike protein

作　　者：郭悦 安柏霖 刘丹丹 罗君红 赵康辰[3] 朱小娟[3] 葛以跃[3] 吴宏斌 崔仑标[1,3] Guo Yue;An Bailin;Liu Dandan;Luo Junhong;Zhao Kangchen;Zhu Xiaojuan;Ge Yiyue;Wu Hongbin;Cui Lunbiao(Nanjing Medical University,Nanjing 210029,China;Tianjin International Biomedical Joint Research Institute,College of Pharmacy,Nankai University,Tianjin 300071,China;NHC Key Laboratory of Enteric Pathogen Microbiology,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;Taizhou Medical City Medical Detection Co.Ltd.,Taizhou 225300,China)

机构地区：[1]南京医科大学,210029 [2]南开大学药学院天津国际生物医药联合研究院,300071 [3]江苏省疾病预防控制中心国家卫生健康委员会肠道病原微生物重点实验室,南京210009 [4]泰州医药城医学检验有限公司,泰州225300

出　　处：《国际病毒学杂志》2022年第4期292-297,共6页International Journal of Virology

基　　金：江苏省社会发展项目(BE2019761、BE2020682);省卫健委重点科研项目(ZD2021060)。

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。Objective 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.

关 键 词：RT-RAA SARS-CoV-2 棘突蛋白 突变 分子检测 

分 类 号：R440[医药卫生—诊断学] O657.3[医药卫生—临床医学]