基于RAA-CRISPR的新型冠状病毒变异位点快速检测方法的建立

Establishment of a rapid detection method for the mutation site of the SARS-CoV-2 based on RAA-CRISPR

目的利用逆转录酶重组酶介导的等温扩增(RT-RAA)与成簇的规律间隔的短回文重复序列(CRISPR核酸检测技术结合,并基于“消线法”CRISPR试纸,建立一种针对新型冠状病毒基因变异位点的现场快速检测方法。方法通过对新型冠状病毒基因组HV69-70del变异位点的序列分析,设计并筛选可高效扩增该变异位点的RT-RAA引物,以及可特异性识别HV69-70del变异位点的crRNA。利用RT-RAA等温扩增技术对目标核酸进行扩增,通过CRISPR-Cas13a系统对扩增产物进行检测并用ERASE试纸进行结果判读。结果该法可特异性区分含有HV69-70del变异位点与未突变的新型冠状病毒核酸,同时在不依赖荧光检测设备的情况下,可在1 h内检出含量为1拷贝/μl的新型冠状病毒HV69-70del变异位点,且与其他7种病原体核酸无交叉反应。结论基于RT-RAA恒温扩增技术以及“消线法”CRISPR检测试纸建立了一种不依赖荧光检测设备的新型冠状病毒变异位点快速检测方法——RAA-CRISPR,为新型冠状病毒变异株的快速检测与鉴定提供了新方法。

Objective To establish a rapid and on-site detection method for SRAS-CoV-2 mutation sites using reverse transcription recombinase aided amplification(RT-RAA)and clustered regularly interspaced short palindromic repeats(CRISPR)nucleic acid detection technology combined with easy-readout and sensitive enhanced(“ERASE”)CRISPR strip.Methods Based on sequence analysis of SRAS-CoV-2 HV69-70del mutation sites,RT-RAA primers for highly efficient amplification of HV69-70del mutation nucleic acid and crRNA(CRISPR RNA)for specific recognition of the HV69-70del site were designed and screened.The target nucleic acid was amplified by RT-RAA technology,the products were detected by the CRISPR-Cas13a system,and the results were interpreted by the“ERASE”strip.Results The detection sensitivity of this method was 1 copy/μl and the HV69-70del mutation site could be specifically identified within 1 h without reliance on fluorescence detection equipment.Then,this method did not cross-react with the other 7 pathogen nucleic acids.Conclusion Based on RT-RAA technology and“ERASE”CRISPR strip technology,a rapid detection method has been established for the HV69-70del mutation site of SRAS-CoV-2——RAA-CRISPR,which does not rely on fluorescence detection equipment and is a new method for rapid detection and identification of SRAS-CoV-2 variants.