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Journal of Integrative Agriculture  2024, Vol. 23 Issue (6): 2149-2153    DOI: 10.1016/j.jia.2024.03.050
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A rapid and visual detection method for Crimean-Congo hemorrhagic fever virus by targeting S gene

Xingqi Liu1*, Zengguo Cao2*, Boyi Li1*, Pei Huang1, Yujie Bai1, Jingbo Huang1, Zanheng Huang1, Yuanyuan Zhang1, Yuanyuan Li1, Haili Zhang1#, Hualei Wang1#

1 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China

2 Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China

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摘要  为建立针对克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus, CCHFV)的简单、快速、适用于现地使用的核酸检测方法,本研究首先比对了20株来自不同国家不同时间分离的CCHFV毒株基因序列,筛选出保守的S基因作为检测靶标,并以中国分离株(YL16070)的S基因保守序列为模板,设计、合成和筛选出反转录重组聚合酶介导恒温扩增(RT-RAA)特异性引物和探针。为实现检测结果的可视化,将四氢呋喃(THF)取代探针3' 端附近的胸腺嘧啶,在探针3' 端引入了阻断基团三碳间隔(C3 spacer),同时对探针和下游引物的5' 端分别使用FAM和生物素标记,最后结合胶体金免疫层析试纸,建立了一种针对CCHFV S基因的可视化快速检测方法CCHFV RT-RAA-VF。为了提高方法的敏感性,对反应温度、反应时间和引物浓度进行了优化,结果显示该方法以浓度为10μM的引物在42 ℃恒温条件下扩增30 min敏感性最高,最低可检出20 copies μL-1的阳性质粒和2 copies μL-1CCHFV RNA转录本。克里米亚-刚果出血热症状与许多出血热病毒感染症状类似,因此将CCHFV与其他可引起出血热的病毒区分开来非常重要。为了评估所建立方法的特异性,分别检测了登革热病毒(DENV)、日本脑炎病毒JEV)、埃博拉病毒(EBOV)、亨德拉病毒(HeV)、尼帕病毒(NIV)及CCHFV的核酸结果表明,该方法仅能识别 CCHFV的核酸,与其他病原无交叉反应。最后为评估该方法的准确性和临床适用性,本研究将所建立的方法与已有的RT-qPCR方法对模拟样品进行平行检测,结果显示两者检测符合率为100%。综上表明,本研究建立的CCHFV RT-RAA-VF快速检测方法敏感性高、特异性好、准确性高,且操作简单、无需特殊仪器,可应用于野外或基层单位CCHFV快速筛查检测工作,在一定程度上响应了世界卫生组织在2018年提出的CCHF研究路线图草案。

Received: 09 September 2023   Accepted: 01 February 2024
Fund: This study was supported by the National Key Research and Development Program of China (2021YFF0703600).
About author:  Xingqi Liu, E-mail: 512793303@qq.com; Zengguo Cao, E-mail: caozg@wh.iov.cn; Boyi Li, E-mail: 2337491685@qq.com; #Correspondence Hualei Wang, E-mail: wanghualei@jlu.edu.cn; Haili Zhang, E-mail: zhanghaili@jlu.edu.cn * These authors contributed equally to this study.

Cite this article: 

Xingqi Liu, Zengguo Cao, Boyi Li, Pei Huang, Yujie Bai, Jingbo Huang, Zanheng Huang, Yuanyuan Zhang, Yuanyuan Li, Haili Zhang, Hualei Wang. 2024.

A rapid and visual detection method for Crimean-Congo hemorrhagic fever virus by targeting S gene . Journal of Integrative Agriculture, 23(6): 2149-2153.

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