寨卡病毒RNA恒温快速扩增检测方法的建立  

Development of an Isothermal Rapid⁃Amplification Detection Method for the RNA of the Zika Virus

作　　者：朱禹 李阿茜 刘洋[2] 李乃哲 余东阳 李川[2] 李建东[2] 王世文[2] 李德新[2] 梁米芳[2] 柳燕 ZHU Yu;LI Aqian;LIU Yang;LI Naizhe;YU Dongyang;LI Chuan;LI Jiandong;WANG Shiwen;LI Dexin;LIANG Mifang;LIU Yan(Department of Microbiology,School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]安徽医科大学基础医学院微生物学教研室,合肥230032 [2]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《病毒学报》2020年第4期610-616,共7页Chinese Journal of Virology

基　　金：国家科技重大专项课题(项目号:2018ZX10711001),题目:病毒感染高通量快速检测与应急筛检技术研究。

摘　　要：2015年以来,寨卡病毒(Zika virus,ZIKV)感染被报告和小头症之间存有关联,已有多个国家和地区报告ZIKV感染证据,我国持续面对ZIKV输入风险。为发展适用于现场的快速高效的ZIKV核酸检测方法,选取ZIKV NS1片段保守区设计特异性引物探针,经优化建立了多种功能蛋白介导的ZIKV RNA恒温快速扩增检测方法。利用所制备的ZIKV NS1片段体外转录RNA作为参考品,并与实时荧光定量RT-PCR检测结果进行比较。同时利用ZIKV细胞培养物中加入健康人血清制备的模拟临床标本进行初步验证。结果显示,建立的RNA恒温快速扩增检测方法可100%检出100拷贝/μL体外转录RNA,可在15 min以内判读,大大低于PCR的常规检测时间;用该方法检测其他相关病毒,无交叉反应。病毒滴度在(104~107 PFU/mL)之间的模拟临床病人标本可达到100%检出。本研究建立的ZIKV RNA恒温扩增快速检测方法快速、灵敏、特异,适用于ZIKV现场应急检测,为ZIKV的防控提供了重要的技术支撑。Since 2015,an association between Zika virus(ZIKV)infection and microcephaly has been reported.ZIKV infection has been documented in several countries and regions,and China continues to suffer the risk of ZIKV importation.We wished to develop a rapid and efficient detection method for the nucleic acids of the ZIKV.Specific primers and probes were designed targeting the conserved region of ZIKV NS1.Various functional proteins involved in our isothermal rapid-amplification method for detection of ZIKV RNA were optimized.ZIKV RNA transcribed in vitro was prepared and used as the standard,and the results were compared with that of real-time quantitative reverse transcription-polymerase chain reaction.Simulated clinical samples were prepared by adding ZIKV cell cultures into normal human serum,and used for preliminary verification.Our amplification method could detect 100%of 100 copies/μL transcribed RNA in vitro within 15 mins,which is much shorter than the conventional detection time of polymerase chain reactions,and cross-reactivity with other related viruses was not observed.For simulated clinical samples,the virus titer was 104–107 PFU/mL,and 100%could be detected.In conclusion,an isothermal amplification detection method for ZIKV RNA was established that proved to be rapid,specific and sensitive.Our method could be applied for emergency detection of the ZIKV,and provide important technical support for the prevention and control ZIKV infection.

关 键 词：寨卡病毒(ZIKV) RNA恒温快速扩增 核酸检测 

分 类 号：R373.1[医药卫生—病原生物学]