一种汉滩病毒和汉城病毒双重实时荧光定量RT-PCR检测方法的建立

目的 建立一种汉滩病毒(Hantaan virus,HTNV)和汉城病毒(Seoul virus,SEOV)双重实时定量荧光RT-PCR检测方法。方法 根据HTNV和SEOV S基因设计引物和探针、优化反应条件,建立2种汉坦病毒的双重实时荧光定量RT-PCR方法。以甲型流感病毒、登革热病毒、新布尼亚病毒、寨卡病毒、新冠病毒阳性核酸为模板验证方法的特异性,将建立的方法与巢氏RT-PCR比较,确定方法对临床样本的适用性。结果 建立了一种HTNV和SEOV 双重实时定量荧光RT-PCR检测方法,该方法对2种型别病毒的最低检测限均为10 copies/μL,不同浓度标准品Ct值批内和批间差异均小于2%。与登革热病毒、新布尼亚病毒、寨卡病毒、新型冠状病毒、甲型流感病毒均无交叉反应。对10份肾综合征出血热急性期血清样本进行检测,结果9份为HTNV、1份为SEOV,与巢式RT-PCR方法结果一致。结论 建立的双重实时荧光定量RT-PCR方法可以快速对HTNV和SEOV 进行分型和定量检测,适用于肾综合征出血热临床早期诊断。

A duplex quantitative real-time RT-PCR assay for the identification of Hantaan and Seoul viruses

To establish a duplex quantitative real-time RT-PCR assay, specific primers and probes were designed using the S gene of Hantaan virus (HTNV) and Seoul virus (SEOV). The real-time RT-PCR reaction conditions were optimized and the sensitivity, specificity, and stability of the method were evaluated. The sensitivity of the method for simultaneous quantitative amplification was 10 copies/μL. The coefficient of variation for both intra- and inter-assays was less than 2%. No cross-reactivity was detected with influenza A virus, dengue virus, severe fever with thrombocytopenia syndrome virus, Zika virus, or SARS-Cov-2. Ten serum samples from HFRS patients were analyzed by this assay and the results showed that nine were HTNVs and one was SEOV, which was consistent with the nested RT-PCR method. Thus, the assay developed in this study was able to detect HTNV and SEOV with good specificity and sensitivity and may become a powerful diagnostic tool, especially in the early stage of HTNV and SEOV infections.