寨卡病毒、登革病毒、基孔肯雅病毒三重荧光定量RT-PCR检测方法的建立及评价  

Development and Evaluation of the Detection Method of Triplex Real-time RT-PCR Assays for Zika,Chikungunya and Dengue Viruses

作　　者：龚雪蕊 李阿茜 刘洋[1] 李川[1] 张全福[1] 李德新[1] 梁米芳[1] 王世文[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室

出　　处：《病毒学报》2018年第1期52-58,共7页Chinese Journal of Virology

基　　金：科技改革发展专项(项目号:国科发资【2016)】273号),题目:寨卡疫情防控科技攻关应急专项~~

摘　　要：本研究旨在建立寨卡病毒（ZIKV virus,ZIKV）、登革病毒（Dengue virus,DENV）以及基孔肯雅病毒（Chikungunya virus,CHIKV）三种病毒快速筛查、诊断的核酸检测技术。选用ZIKV的NS1基因、DENV的NS5蛋白基因以及CHIKV的E1蛋白基因作为靶标区域设计三组特异性引物探针,建立三重实时荧光定量RT-PCR检测方法。用ZIKV、DENV、CHIKV病毒体外转录RNA和病毒细胞培养物对该方法的灵敏性、特异性、重复性等方面进行评价,最后临床样本以及模拟标本验证。结果显示：三重实时荧光定量RT-PCR检测方法扩增效率均可达到90%以上,三种病毒体外转录RNA最低检测限均低于15拷贝/PCR,病毒培养物最低检出限均低于10PFU/mL且与单重检测方法无明显差异。与其他病毒无交叉反应,变异系数均在2%以内。临床标本及模拟标本检出率均可达95%以上。本研究建立的检测寨卡病毒、登革病毒以及基孔肯雅病毒的三重实时荧光RT-PCR方法具有良好的敏感性、特异性和重复性,可用于寨卡病毒病等相关临床标本的检测。To achieve rapid nucleic acid detection and diagnosis of Zika virus（ZIKA）,Dengue virus（DENV）and Chikungunya virus（CHIKV）infection,the primers and probes targeting the ZIKV NS1 gene,DENV NS5 gene and CHIKV E1 gene were designed,and then a triplex Real-time RT-PCR assay was developed.The specificity,sensitivity and stability were evaluated using in vitro transcribed RNA and virus isolate of ZIKV,DENV and CHIKV,followed by clinical specimen verification.As results showed,for the three viruses detection,the amplification efficiency of the triplex assay were all above 90% .The limit of detection（LOD）of in vitro transcribed RNAs were less than 15 Copies/PCR,and the LOD of three virus isolates were lower than 10 PFU/mL.Compared with related monoplex assays,there was no significant difference.There was no cross reaction with other virus,and variable coefficient of CT value were all less than 2% .In 20 serum samples of dengue acute-phase patients,19 were detected positive.All of 50 healthy serum samples were detected negative.In conclusion,a triplex real-time RT-PCR assay for ZIKV,DENV and CHIKV detection was established,and proved to be specific and sensitive.So this test method can be used for laboratory detection of suspected Zika virus disease and other related cases.

关 键 词：三重实时荧光定量RT-PCR 寨卡病毒(ZIKV) 登革病毒(DENV) 基孔肯雅病毒(CHIKV) 

分 类 号：R392[医药卫生—免疫学]