双重荧光RT-RAA检测5种蚊媒病毒方法的建立

Development of the detection for five mosquito-borne viruses based on duplex fluorescent RT-RAA assay

目的建立快速筛查登革病毒(DENV)、乙型脑炎病毒(JEV)、寨卡病毒(ZIKV)、黄热病毒(YFV)以及基孔肯雅病毒(CHIKV)5种蚊媒病毒的重组酶介导等温扩增(RAA)方法。方法分析上述5种病毒的保守基因作为靶标区域并引入人核糖核酸酶P(RNaseP)基因作为内对照,设计6组特异性引物和探针,并根据引物二聚体和错配等生物信息学分析将6组引物和探针组合成3组双重荧光RT-RAA检测体系,在相同反应条件下进行检测。用5种病毒的体外转录RNA验证3组荧光RT-RAA检测体系的灵敏度,用疟原虫、汉坦病毒、汉城病毒以及流感病毒核酸验证方法的特异性;分别用中浓度(10^(4)拷贝/μl)、低浓度(10^(2)拷贝/μl)5种病毒的体外转录RNA验证3组双重荧光RT-RAA检测体系的重复性。结果通过生物信息学分析,将5种病毒和RNaseP组合为3组双重荧光RT-RAA检测体系,分别为DENV与ZIKV、JEV与YFV以及CHIKV与RNaseP,均在39℃反应15 min。3组双重荧光RT-RAA检测体系检测5种病毒体外转录RNA的灵敏度介于1~10^(2)拷贝/μl,其中检测DENV、ZIKV和YFV的灵敏度均为1拷贝/μl,JEV灵敏度为10拷贝/μl,CHIKV灵敏度为10^(2)拷贝/μl;3组荧光RT-RAA检测体系均与其他病毒无交叉反应。5种病毒的中浓度体外转录RNA的检出率均达到100.0%,低浓度体外转录RNA也能达到83.3%。结论本研究建立的检测方法具有良好的敏感性、特异性和重复性,可用于人体及蚊类样本中5种蚊媒病毒的筛查。

Objective To establish a detection method for Dengue virus(DENV),Japanese encephalitis virus(JEV),Zika virus(ZIKV),Yellow fever virus(YFV)and Chikungunya virus(CHIKV)with recombinase aided amplification(RAA).Methods The conserved genes of the above 5 viruses were analyzed and determined as the target regions,and human ribonuclease P gene(RNaseP)was introduced as the internal control.Six groups of specific primers and probes were designed.According to the bioinformatics analysis of primer dimer and mismatch,the six groups of primers and probe were combined into three groups of dual fluorescent RT-RAA which could be performed under the same reaction conditions.The sensitivity of the three group reactions was verified by using the in vitro transcript RNA of the five virus target genes.The specificity was verified by using the nucleic acid of Plasmodium,Hantavirus,Seoul virus and Influenza viruses.The in vitro transcript RNA of the medium concentration(10^(4)copies/μl)and low concentration(10^(2)copies/μl)was verified for the repeatability of three groups of dual fluorescent RT-RAA assay.Results Through bioinformatics analysis,five viruses and RNaseP genes were combined into three groups of dual fluorescent RT-RAA reactions,DENV and ZIKV,JEV and YFV,CHIKV and RNaseP,respectively.The results can be obtained under 39℃for 15 minutes.Three groups of dual fluorescent RT-RAA assay were used to detect the in vitro transcript RNA of five viruses,the sensitivity was between 1-10^(2)copies/μl.The sensitivity of DENV,ZIKV and YFV was 1 copy/μl.JEV was 10 copies/μl,CHIKV was 10^(2)copies/μl.The three groups of fluorescent RT-RAA assay had no cross reaction with other viruses.The positive rate of medium concentration of in vitro transcript RNA of the five viruses was 100.0%,and the positive rate of low concentration of in vitro transcript RNA was 83.3%.Conclusion The assay established in this study has good sensitivity,specificity and repeatability,it can be used for the differentiation of DENV,JEV,ZIKV,YFV and CHIKV.