新型冠状病毒双重荧光RT-PCR检测方法

   杨宇1，刘丽娟1，赵婷婷1，李辉2，王莎莎1，徐焕州1，杨鹏飞1，王旺1，孙肖红1，王静1

    1中国检验检疫科学研究院卫生检疫研究所，北京100123；2.上海辉睿生物科技有限公司

    摘要：目的为快速检测新型冠状病毒，防控疫情的输入，建立一种快速的双重实时荧光RT-PCR检测方法。方法对WHO公布的单重实时荧光RT-PCR检测引物和探针进行重新设计和优化，建立双重荧光RT-PCR反应体系，分别采用羧基荧光素（FAM）和绿色荧光蛋白（VIC）荧光基团标记探针，实现双基因的同时检测。结果经优化的双重实时荧光RT-PCR方法有较好的灵敏度和特异性，对阳性对照质粒检测灵敏度为31拷贝/μl，检测健康和普通发热人员咽拭子以及流感病毒无交叉反应。结论建立了双重实时荧光RT-PCR快速检测方法，提高检测效率和准确度的同时降低了成本，可用于新型冠状病毒的快速应急检测。

    关键词：实时荧光PCR；新型冠状病毒；检测；交叉反应

    中图分类号：R183.4　文献标识码：B

    Multiplex Real-time PCR method for rapiddetection of

    Novel Coronavirus

    YANG Yu*, LIU Li-juan, ZHAO Ting-ting, LIHui, WANG Sha-sha, XU Huan-zhou,

    YANG Peng-fei, WANG wang, SUN Xiao-hong, WANGJing

    *Institute of Health Quarantine, ChineseAcademy Of Inspection and Quarantine, Beijing 100123,China

    Abstract:   Objective  To establish a rapid, sensitive method to detect Novel Coronaviruswhich are acute infections with high case fatality rates bymultiplex real-time fluorescence quantitative RT- PCR. Methods   Taqman probes labeled by FAM and VIC. Themultiplex real-time quantitative RT-PCR assay was optimized basedon the reported mono assay.  The sensitivity was evaluated andinfluenza virus and human swab were using to examine thespecificity.  Results   A specic real-time RT-PCRmethod was developed with the sensitivity of 31copies/μl for NovelCoronavirus, a synthetic plasmid DNA as a positive control，and nocross reaction was found with Influenza virus and human swabsamples.  Conclusion   This multiple assay has goodprospects of application for rapid test.

    Key words:   Multiplex Real-timePCR; Novel Coronavirus; Detection; Cross reaction

    《中国国境卫生检疫杂志》2012年第5期