SFTS病毒、登革病毒、汉滩病毒快速荧光定量RT-PCR检测方法的建立及评价  

Development and evaluation of the detection method of rapid RT-PCR assays for severe fever with thrombocytopenia syndrome virus,dengue virus and hantaan virus

作　　者：胡停停 朱禹 李阿茜 孙丽娜[1] 黄晓霞[1] 芜为[1] 李川[1] 王芹[1] 李建东[1] 李德新[1] 王世文[1] 柳燕[2] 梁米芳[1] 王晓芳[1] Hu Tingting;Zhu Yu;Li Aqian;Sun Lina;Huang Xiaoxia;Wu Wei;Li Chuan;Wang Qin;Li Jiandong;Li Dexin;Wang Shiwen;Liu Yan;Liang Mifang;Wang Xiaofang(Key Laboratory of Biosafety,National Health and Family Planning Commission,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Anhui Medical University,Hefei 230032,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所国家卫生健康委生物安全重点实验室,北京102206 [2]安徽医科大学,合肥230032

出　　处：《中华实验和临床病毒学杂志》2022年第2期230-235,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：传染病防治重大专项(2018ZX10711-001,2017ZX10303404-007-002)。

摘　　要：目的建立针对发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)、登革病毒(dengue virus,DENV)、汉滩病毒(hantaan virus,HTNV)三种常见病毒性出血热病毒的现场应急快速检测方法。方法基于传统的TaqMan荧光探针技术,利用国产快速一步法RT-PCR试剂盒,结合magnetic induction cycler(Mic)qPCR仪,建立三种常见病毒性出血热病毒的快速荧光定量RT-PCR检测方法。利用三种病毒体外转录RNA及模拟样本、其他相关病毒临床样本及健康人血标本进行灵敏度、特异性、重复性验证。结果相较于传统的实时荧光定量RT-PCR方法,此方法所需时间大大缩短,可在35 min内完成检测。SFTSV、DENV、HTNV的最低检出限均小于100拷贝/PCR,与模拟对照样本及其他病毒临床样本无交叉反应,模拟阳性样本验证均可检出,且各组验证结果的Ct值变异系数均小于4%。结论本研究建立的快速荧光定量RT-PCR检测方法具有良好的灵敏性、特异性和重复性。对于现场应急检测和媒介生物标本筛查具有一定的应用前景。Objective To establish a quick on-site emergency detection method for severe fever with thrombocytopenia syndrome virus(SFTSV),dengue virus(DENV),and hantaan virus(HTNV).Methods This research was based on the traditional TaqMan fluorescent probe technology,using the domestic rapid one-step quantitative RT-PCR kit,combined with the Magnetic induction cycler(Mic)qPCR instrument.The detection limit,specificity and repeatability of this method were evaluated by simulated samples,other virus infected samples and normal human blood samples.Results Compared with the traditional RT-PCR assay,the required time of this method was greatly shortened,and the detection can be completed within 35 minutes.The limit of quantitation for SFTSV,DENV and HTNV are less than 100copies/PCR.No nonspecific amplification was found in the simulated negative samples and other virus infected samples.All the simulated positive sample for verification could be detected,and coefficient of variation Ct value of each group was less than 4%.Conclusions The rapid fluorescence quantitative RT-PCR assays have certain application prospects for on-site emergency detection,and provide important technical supports and new directions for the prevention and control of common hemorrhagic fever viruses.

关 键 词：发热伴血小板减少综合征病毒 登革病毒 汉滩病毒 快速荧光定量RT-PCR 

分 类 号：R512.8[医药卫生—内科学] R440[医药卫生—临床医学]