重组酶聚合酶侧流层析技术检测新冠病毒核酸快速诊断方法的初步建立

Preliminary establishment of a rapid diagnosis method for detecting SARS-CoV-2 nucleic acid through recombinase polymerase amplification combined with a lateral flow dipstick

目的建立重组酶聚合酶扩增侧流层析技术(RPA-LFD)检测新冠病毒(SARS-CoV-2)核酸快速诊断方法,为基层医院和边远地区防控新冠病毒疫情提供技术支持。方法根据新冠病毒N基因核苷酸序列中保守序列,通过生物信息学软件分析、设计和筛选最佳的重组酶聚合酶扩增引物及侧流层试纸探针,优化重组酶聚合酶扩增反应条件以及检验建立方法的灵敏性和特异性。结果利用RPA-LFD法在37℃下反应15 min可检测到每个反应最低100 fg含量的新冠病毒,并与流感病毒、副流感病毒、鼻病毒和腺病毒没有交叉反应,因此据此可以建立灵敏性高、特异性强和可操作性强的新冠病毒快速诊断方法。结论通过RPA-LFD技术建立的新冠病毒快速诊断方法可获得较为理想的灵敏度、特异性,为进一步研制新冠病毒核酸快速诊断试剂盒奠定了基础。

This study aimed to develop a rapid diagnostic method for the detection of SARS-CoV-2 nucleic acid based on recombinase polymerase amplification combined with a lateral flow dipstick(RPA-LFD),to provide technical support for the prevention and control of SARS-CoV-2 epidemics in basic hospitals and remote areas.According to the conserved nucleotide sequence of the N gene of SARS-CoV-2,the best recombinase polymerase amplification primers and lateral flow strip probes were designed,analyzed and screened in bioinformatics software.The reaction conditions for recombinase polymerase amplification were optimized,and the sensitivity and specificity of the established method were examined.The RPA-LFD assay for detecting SARS-CoV-2 performed best at 37℃at 15 min.The lowest quantity of SARS-CoV-2 detected in a reaction was 100 fg.No cross-reaction with influenza virus,parainfluenza virus,rhinovirus and adenovirus in the RPA-LFD assay was found.Thus,an easily performed,rapid diagnostic method for detecting SARS-CoV-2 with high sensitivity and specificity was established.In conclusion,our preliminary rapid diagnostic method for detecting SARS-CoV-2 with good sensitivity and specificity through RPA-LFD is worthy of further clinical application.