中国媒介生物学及控制杂志 ›› 2023, Vol. 34 ›› Issue (3): 285-290.DOI: 10.11853/j.issn.1003.8280.2023.03.001

• 实验研究 •    下一篇

流行性乙型脑炎和西尼罗病毒双重微滴数字PCR检测方法的建立

张俊锋1,2, 张雅丽2,3, 王瑞晨2, 禄阳2, 张天姿2, 付士红2, 殷启凯2, 李樊2, 何英2, 聂凯2, 马超锋3,4, 梁国栋2, 扈瑞平1, 许松涛2, 王环宇2   

  1. 1. 内蒙古医科大学基础医学院生物化学与分子生物学教研室, 内蒙古 呼和浩特 010110;
    2. 中国疾病预防控制中心病毒病预防控制所虫媒病毒室, 北京 102206;
    3. 陕西中医药大学公共卫生学院, 陕西 咸阳 712360;
    4. 西安市疾病预防控制中心, 陕西 西安 710000
  • 收稿日期:2023-01-18 出版日期:2023-06-20 发布日期:2023-06-16
  • 通讯作者: 扈瑞平,E-mail:783674348@qq.com;许松涛,E-mail:xust@ivdc.chinacdc.cn
  • 作者简介:张俊锋,女,在读硕士,主要从事病原检测方法建立,E-mail:2384999538@qq.com
  • 基金资助:
    美国NIH项目U01(AI151810)

Establishment of a duplex droplet digital PCR assay for Japanese encephalitis and West Nile viruses

ZHANG Jun-feng1,2, ZHANG Ya-li2,3, WANG Rui-chen2, LU Yang2, ZHANG Tian-zi2, FU Shi-hong2, YIN Qi-kai2, LI Fan2, HE Ying2, NIE Kai2, MA Chao-feng3,4, LIANG Guo-dong2, HU Rui-ping1, XU Song-tao2, WANG Huan-yu2   

  1. 1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Inner Mongolia Medical University, Hohhot, Inner Mongolia 010110, China;
    2. Department of Arbovirus, Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
    3. School of Public Health, Shaanxi University of Traditional Chinese Medicine, Xianyang, Shaanxi 712360, China;
    4. Xi'an Center for Disease Control and Prevention, Xi'an, Shaanxi 710000, China
  • Received:2023-01-18 Online:2023-06-20 Published:2023-06-16
  • Supported by:
    US NIH Project U01 (No. AI151810)

摘要: 目的 建立流行性乙型脑炎病毒(JEV)和西尼罗病毒(WNV)的双重微滴式数字PCR(ddPCR)检测方法。方法 基于已设计的JEV和WNV引物探针,建立JEV和WNV双重ddPCR检测反应体系,摸索检测的敏感性、特异性和可重复性,灵敏度与双重荧光定量PCR(qPCR)的每个反应管内荧光信号达到设定的阈值所经历的循环数(Ct)值做对比。结果 双重ddPCR检测反应体系对JEV和WNV的检测灵敏度均可达到102拷贝/μl,该方法的可重复性、特异性良好,未发现与登革病毒、基孔肯雅病毒、寨卡病毒、蜱传脑炎病毒以及人类基因组有交叉反应。结论 建立的双重ddPCR方法能敏感、特异检测JEV和WNV,为不同场景下这2种病毒的检测提供了解决方案。

关键词: 流行性乙型脑炎病毒, 西尼罗病毒, 微滴式数字聚合酶链式反应

Abstract: Objective To establish a duplex droplet digital polymerase chain reaction (ddPCR) detection method for Japanese encephalitis virus (JEV) and West Nile virus (WNV).Methods Based on the designed primers and probes of JEV and WNV, a duplex ddPCR detection system for JEV and WNV was established. Its sensitivity, specificity, and repeatability were explored. The sensitivity was compared with the number of cycles required for the fluorescent signal to cross the threshold in each reaction tube of dual quantitative PCR.Results The detection sensitivity of the duplex ddPCR detection system could reach 102 copies/μl for both JEV and WNV, with good specificity and repeatability. No cross-reactivity was observed with the Dengue virus, Chikungunya virus, Zika virus, Tick-borne encephalitis virus, and human genome.Conclusion The established duplex ddPCR method shows high sensitivity and specificity for JEV and WNV detection, which provides a solution for detection for the two viruses in different scenarios.

Key words: Japanese encephalitis virus, West Nile virus, Droplet digital polymerase chain reaction

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