论文部分内容阅读
目的:建立一种实时荧光逆转录重组酶介导的等温扩增（reverse transcription recombinase-aided amplification，RT-RAA）技术检测寨卡病毒（Zika virus，ZIKV）的方法，以实现ZIKV的现场快速筛查及诊断。方法:通过基因组序列比对，选择ZIKV保守序列设计RT-RAA特异性引物和探针，并用系列稀释的ZIKV重组质粒与毒株核酸验证方法的灵敏性与重复性，通过检测登革病毒、基孔肯雅病毒、西尼罗病毒等其他虫媒病毒验证方法的特异性。结果:建立的RT-RAA方法可在39 ℃恒温条件下30 min内对ZIKV进行高效扩增，检测系列稀释的ZIKV重组质粒，其95%检测限可达到15拷贝/反应，特异性强，与登革病毒、基孔肯雅病毒、西尼罗病毒虫媒病毒无交叉反应，且重复性好。结论:本研究建立的ZIKV的RT-RAA等温扩增方法，具有反应迅速，无需精密仪器，操作简单等优点，适合于应急快速检测。“,”Objective:To establish a real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) technology to detect Zika virus (ZIKV) for rapid on-site screening and diagnosis of ZIKV.Methods:Through genomic sequence alignment, conserved sequences of ZIKV were selected to design RT-RAA specific primers and probes. Serial dilutions of ZIKV recombinant plasmids and strain nucleic acids were used to verify the sensitivity and reproducibility of this method. The specificity was evaluated by testing of other arboviruses, including dengue virus, chikungunya virus and West Nile virus.Results:The established RT-RAA method can efficiently amplify ZIKV in 30 minutes at a constant temperature of 39℃. For the detection of serially diluted ZIKV recombinant plasmids, the 95% detection limit can reach 15 copies/reaction. The assay showed strong specificity, and there was no cross-reaction with other arbovirus, including dengue virus, chikungunya virus and West Nile virus. The reproducibility of the method was also good.Conclusions:The isothermal amplification method of RT-RAA for ZIKV established in this study has the advantages of rapid response, no need for precision instruments, simple operation, etc., and is suitable for emergency rapid detection.