摘要
建立寨卡病毒(Zika virus,ZIKV)、登革病毒(Dengue virus,DENV)和黄热病毒(Yellow fever virus,YFV)3种蚊媒传染病核酸快速实时荧光定量检测方法。以寨卡病毒polyprotein gene基因,登革病毒NS5基因,黄热病毒3’UTR基因作为靶区域设计相应的引物探针,探针的5’端分别标记不同的荧光基团,3’端标记相应的淬灭基团,对扩增体系中引物探针浓度、热启动Taq酶浓度、逆转录酶浓度、RNA酶抑制剂浓度分别进行优化。构建了一种可同时用于寨卡病毒、登革病毒和黄热病毒三重快速荧光PCR检测体系,25μL扩增体系中ZIKV、DENV和YFV的引物浓度分别为200 nmol/L、240 nmol/L和240 nmol/L;探针浓度分别为200 nmol/L、240 nmol/L和80 nmol/L;热启动Taq酶、逆转录酶和RNA酶抑制剂的终浓度为分别为2.5U/反应、60U/反应和80U/反应。该检测体系对3种病原体的最低检测限均为1.0×10^(3)拷贝/mL,在MIC IAT C2-48和ABI7500两种不同型号的实时荧光定量PCR仪上检测结果一致,对登革热、寨卡和黄热病毒血清样本检测均为阳性扩增,与丙型肝炎病毒、乙型脑炎病毒、副流感病毒、腺病毒等无交叉反应。因此,本研究建立的寨卡病毒、登革病毒和黄热病毒的三重快速荧光PCR检测体系可用于对上述3种病毒的定性或定量检测。
To create a rapid nucleic-acid diagnostic method for the Zika virus(ZIKV),Dengue virus(DENV)and Yellow fever virus(YFV).Specific primers and probes based on the polyprotein gene of the ZIKV,NS5 gene of the DENV,and 3'untranslated region(UTR)gene of the YFV as target areas were designed.The 5'ends of probes were marked with different fluorescent groups.The 3'ends were marked with the corresponding quenching groups.A rapid tri-plex quantitative polymerase chain reaction(qPCR)detection method was developed for ZIKV,DENV and YFV by optimizing the concentration of the primers and probes,Taq DNA polymerase,reverse transcriptase and RNA inhibitor.The primer concentration(nmol/L)of the ZIKV,DENV and YFV in a 25-μL amplification system was 200,240 and 240,and the probe concentration(nmol/L)was 200,240 and 80,respectively.The final concentration(in U/reaction)of Hi-Taq polymerase,M-MLV and RNA inhibitors was 2.5,60 and 80,respectively.The limit of detection for the three pathogens was 1.0X10 copy/mL.These results were consistent with those achieved using MIC IAT C2-48 and ABI 7500 systems.and there was no cross-reaction with the hepatitis C virus,B encephalitis virus,parainfluenza virus or adenovirus.Therefore,our triple-fast fluorescence PCR detection system could be used for simultaneously detection of the ZIKV,DENV and YFV.
作者
徐聪灵
王静
杨绪理
陈峰
刘文干
耿合员
XU Congling;WANG Jing;YANG Xuli;CHEN Feng;LIU Wengan;GENG Heyuan(Lianyungang Customs,Lianyungang 22042,China;Nantong Customs,Nantong 226000,China;Anhui Anlong Gene Technology Co.Ltd,Hefei 230000,China;Jiangsu International Travel Health Care Center(Nanjing Customs Port Outpatient Department),Nanjing 210019,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2023年第3期743-751,共9页
Chinese Journal of Virology
基金
连云港海关(原连云港出入境检验检疫局自立项课题)(项目号:LYG2017KJ06),题目:基于微流控荧光PCR技术的重要蚊媒传染病检测试剂的研究。
关键词
快速荧光PCR检测
蚊媒病毒
Rapid fluorescence PCR detection
Mosquito-borne viruses
