检测寨卡病毒核酸的RT-LAMP可视化方法的建立

Avisual RT-LAMP-VF method for detection of Zika virus nucleic acid

为建立简单、便捷、灵敏、特异的寨卡病毒(Zika virus,ZIKV)快速检测方法,本研究通过对不同时间、不同地区分离的ZIKV全基因组序列进行分析,筛选ZIKV NS5基因保守区为靶标,同时设计特异性引物及探针,结合RT-LAMP恒温扩增技术和免疫层析技术,成功构建ZIKV反转录环介导等温扩增核酸可视化(RT-LAMP-VF)检测方法。结果显示,该方法具有良好的特异性和敏感性,当内、外、环引物比为FIP∶LF∶F3=4∶2∶1时,在61℃条件下恒温扩增45 min,可特异性检测到10^(2)copies/μL的RNA标准品以及2.15 pfu的ZIKV,且与其他黄病毒(日本乙型脑炎病毒、猪瘟病毒)等无交叉反应。血液组织中的其他RNA不影响RT-LAMP-VF的敏感性和特异性,表明该方法可用于临床样本ZIKV的检测。本研究建立的ZIKV RT-LAMP-VF检测方法操作简单、不需要特殊仪器设备,尤其适用于基层单位ZIKV的现场快速检测,为ZIKV病的现场快速检测和预警预报体系的建立提供技术支持与物质保障。

To establish a simple,convenient,sensitive,and specific method for rapid detection of Zika virus(ZIKV),the whole genome sequences of ZIKV isolated from different times and regions were analyzed.The specific primers and probes were designed based on the screened target sequences located in the conserved region of the ZIKV NS5 gene.By combining RT-LAMP isothermal amplification technology and immunochromatography technology,a reverse transcription loop mediated isothermal amplification nucleic acid and flow visualization strip(RT-LAMP-VF)detection method for ZIKV was established.The results showed that the method had good specificity and sensitivity.When the ratio of inner,outer,and ring primers(FIP∶LF∶F3)was 4∶2∶1,the detection method can specifically detect 10^(2)copies/μL RNA transcripts or 2.15 pfu ZIKV at 61℃for 45 minutes,with no cross reaction with other flaviviruses such as Japanese encephalitis virus and classical swine fever virus.Other RNAs in blood tissue samples did not affect the sensitivity and specificity of RT-LAMP-VF,indicating that the method can be applied to clinical practice.The ZIKV RT-LAMP-VF detection method established in this study is easy to perform and does not require special instruments and equipment.It is particularly suitable for the rapid detection of ZIKV in grassroots units,providing technical support and material support for the establishment of on-site rapid detection and early warning and prediction systems for ZIKV disease.