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  

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

作　　者：刘师文[1] 熊英[1] 施勇[1] 李健雄[1] 王倩 龚甜[1] LIU Shi-wen;XIONG Ying;SHI Yong;LI Jian-xiong;WANG Qian;GONG Tian(Jiangxi Center for Disease Control and Prevention,Nanchang 330029,China)

机构地区：[1]江西省疾病预防控制中心,南昌330029

出　　处：《中国人兽共患病学报》2021年第6期478-483,共6页Chinese Journal of Zoonoses

基　　金：江西省科技计划项目重点研发计划(No.20202BBGL73052)。

摘　　要：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。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.

关 键 词：汉滩病毒 汉城病毒 双重实时荧光定量RT-PCR 

分 类 号：R373[医药卫生—病原生物学] R331[医药卫生—基础医学]