流行性乙型脑炎病毒、西尼罗病毒、黄热病毒和寨卡病毒四重LuminexxTAG检测方法的建立与应用  

Development and Application of Quadruple Luminex xTAG Method for Simultaneous Detection of Japanese Encephalitis Virus,West Nile Virus,Yellow Fever Virus and Zika Virus

作　　者：孙俊颖 伍绮文 李吉初 李春红 郭鹏举 黄艺 尹飞飞 SUN Junying;WU Qiwen;LI Jichu;LI Chunhong;GUO Pengju;HUANG Yi;YIN Feifei(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of Animal Disease Control/Guangdong Scientific Observation and Experiment Station of Veterinary Medicine and Diagnostic Technology,Ministry of Agriculture and Rural Affairs,Guangzhou 510640,China;Guangzhou Weibai Biotechnology Co.Ltd.,Guangzhou 510700,China;School of Basic Medicine and Life Sciences,Hainan Medical University,Haikou 571199,China)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [2]广州维佰生物科技股份有限公司,广东广州510700 [3]海南医学院基础医学与生命科学学院,海南海口571199

出　　处：《广东农业科学》2024年第10期78-87,共10页Guangdong Agricultural Sciences

基　　金：国家自然科学基金(82060378);广州市开发区国际科技合作项目(2019GH08);广东省“十四五”农业科技创新十大主攻方向“揭榜挂帅”项目(2022SDZG02);海南省重点研发计划项目(ZDYF2022SHFZ085)。

摘　　要：【目的】黄病毒科虫媒病毒引起的人畜共患传染病对我国畜牧业及公共卫生造成严重影响。虫媒病毒种类多且感染症状相似,临床监测工作繁重,建立4种危害严重的黄病毒科虫媒病毒快速、高通量检测技术为其诊断和流行病学监测提供技术支撑。【方法】利用Luminex xTAG技术,针对流行性乙型脑炎病毒(JEV)5'UTR、西尼罗病毒(WNV)5'UTR及部分C基因、黄热病毒(YFV)5'UTR和寨卡病毒(ZIKV)NS5基因保守区,设计4对特异性引物并对引物进行TAG和Biotin修饰,以标准毒株为模板,进行多重PCR扩增;将扩增产物与带有反向TAG序列的不同编号荧光MagTAG磁珠、链霉亲和素R-藻红蛋白进行核酸杂交反应,利用Luminex 200系统检测磁珠荧光信号和藻红蛋白荧光信号,对病原进行分类和定量检测。【结果】建立了能特异性检测JEV、WNV、YFV和ZIKV的四重Luminex xTAG方法,其最优引物工作浓度为0.5、0.5、0.75、0.5µmol/L;杂交工作体系为磁珠工作液20μL、PCR扩增产物5μL、SAPE报告缓冲液75μL;杂交温度为37℃,杂交时间为30 min,pH值为8.0;该检测方法特异性好且不与登革病毒等其他虫媒病毒发生交叉反应。重复性试验结果表明,Luminex xTAG方法的批内变异系数为2.50%~5.63%,批间变异系数为3.61%~12.50%。四重Luminex xTAG方法对JEV和ZIKV的检测限为1×10^(4) copies/μL,对WNV和YFV的检测限为1×10^(3)copies/μL,其检测WNV、YFV和ZIKV的灵敏度较常规PCR高10~100倍。用Luminex xTAG方法和RT-qPCR方法检测209份临床样品和模拟样品,JEV、WNV、YFV、ZIKV符合率为100%。【结论】建立的四重Luminex xTAG方法具有高通量、高特异性和灵敏度、成本效益高的优点,可为虫媒病毒临床诊断和流行病学监测提供一种高通量技术手段。【Objective】The zoonotic infectious diseases caused by arbovirus of family Flavivirus have a severe impact on China’s animal husbandry and public health.Due to the wide types and similar infection symptoms of arbovirus,as well as its heavy clinical surveillance,the study aims to establish a fast and high-throughput detection technology for four serious Flavivirus arbovirus to provide technical support for clinical diagnosis and epidemiological monitoring of arbovirus.【Method】Based on Luminex xTAG technology,four pairs of specific primers were designed for 5'UTR of Japanese encephalitis virus(JEV),5'UTR and part of C gene of West Nile virus(WNV),5'UTR of yellow fever virus(YFV),NS5 gene of Zika virus(ZIKV),and were modified with TAG sequence and Biotin.Multiplex PCR amplification was carried out with standard virus strains as model.Then,PCR products were hybridized with magnetic beads with complementary TAG sequences and streptavidin-phycoerythrin,and the fluorescence signals of magnetic beads and phycoerythrin were detected by Luminex 200 instrument to indicate the classification and quantification of the pathogens of the arbovirus samples.【Result】The Luminex xTAG method applied to detect JEV,WNV,YFV and ZIKV was established,and the optimal primer working concentration was 0.5,0.5,0.75,0.5µmol/L;the established hybridization system and reaction conditions were:20μL of magnetic bead working solution,5μL of PCR amplification product,and 75μL of SAPE working buffer solution;the hybridization temperature,hybridization time and pH value were 37℃,30 min,and 8.0,respectively.The quadruple Luminex xTAG method could detect JEV,WNV,YFV and ZIKV simultaneously,and there was no cross reaction with dengue virus.The duplicate test results indicated that,the coefficient of variation of the intra-assay for quadruple Luminex xTAG method was 2.50%-5.63%and inter-assay was 3.61%-12.50%.The detection limits of JEV and ZIKV were 1×10^(4)copies/μL,and those of WNV and YFV were 1×10^(3)copies/μL,respectively.The

关 键 词：虫媒病毒 流行性乙型脑炎病毒 西尼罗病毒 黄热病毒 寨卡病毒 液相芯片 LuminexxTAG技术 

分 类 号：S854[农业科学—临床兽医学]