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  

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

作　　者：张俊锋 张雅丽 王瑞晨 禄阳 张天姿 付士红[2] 殷启凯 李樊 何英[2] 聂凯[2] 马超锋 梁国栋[2] 扈瑞平[1] 许松涛 王环宇[2] ZHANG Jun-feng;ZHANG Ya-li;WANG Rui-chen;LU Yang;ZHANG Tian-zi;FU Shi-hong;YIN Qi-kai;LI Fan;HE Ying;NIE Kai;MA Chao-feng;LIANG Guo-dong;HU Rui-ping;XU Song-tao;WANG Huan-yu(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Inner Mongolia Medical University,Hohhot,Inner Mongolia 010110,China;Department of Arbovirus,Institute of Viral Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Beijing 102206,China;School of Public Health,Shaanxi University of Traditional Chinese Medicine,Xianyang,Shaanxi 712360,China;Xi’an Center for Disease Control and Prevention,Xi’an,Shaanxi 710000,China)

机构地区：[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

出　　处：《中国媒介生物学及控制杂志》2023年第3期285-290,共6页Chinese Journal of Vector Biology and Control

基　　金：美国NIH项目U01(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。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.

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 

分 类 号：R373[医药卫生—病原生物学]