登革病毒、黄热病毒和基孔肯雅病毒三重实时荧光定量RT-PCR检测方法的建立及评价  

Development and evaluation of the detection method of triplex real-time fluorescent quantitative RT-PCR assays for dengue,yellow fever and chikungunya viruses

作　　者：阚乃鹏 翁育伟[1] 俞婷婷 王金章[1] Kan Naipeng;Weng Yuwei;Yu Tingting;Wang Jinzhang(Fujian Provincial Center for Disease Control and Prevention,Fuzhou 350012,China;College of Life Sciences,Fujian Normal University,Fuzhou 350108,China)

机构地区：[1]福建省疾病预防控制中心,福州350012 [2]福建师范大学生命科学学院,福州350108

出　　处：《中华实验和临床病毒学杂志》2022年第6期707-711,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：福建省卫生健康科技计划项目(2020GGA024,2020QNA021);福建省科技创新平台建设项目(2019Y2001)。

摘　　要：目的建立登革病毒(dengue virus,DENV)、黄热病毒(yellow fever virus,YFV)和基孔肯雅病毒(chikungunya virus,CHIKV)核酸的三重实时荧光定量逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测方法。方法通过全球共享数据库下载DENV(Ⅰ、Ⅱ、Ⅲ、Ⅳ型)、YFV、CHIKV全基因组序列进行比对分析,针对这3种病毒的相对保守区域分别设计特异性引物和探针,建立三重实时荧光定量RT-PCR检测方法,以其他病毒核酸评价方法的特异性,用病毒体外转录RNA评价方法的灵敏度,以病毒高、中、低浓度核酸进行独立重复实验评价方法的重复性。DENV采用登革热患者血清进行验证,YFV、CHIKV采用模拟阳性标本进行验证,采用健康人血清进行阴性验证。结果本方法与其他病毒核酸无交叉反应;对DENV(Ⅰ、Ⅱ、Ⅲ、Ⅳ型)、YFV、CHIKV的最低检出限分别为21.55拷贝/反应、21.25拷贝/反应、21.85拷贝/反应、22.75拷贝/反应、22拷贝/反应、45.65拷贝/反应;各浓度检测Ct值的标准差在0.5以内、变异系数在3%以下;临床阳性标本及模拟阳性标本的检出率为100%,健康人血清的阴性率为100%。结论本研究建立的DENV、YFV和CHIKV的三重实时荧光定量RT-PCR检测方法具有良好的特异性、灵敏度和重复性。Objective To develop a triplex real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR)assay for dengue virus(DENV),yellow fever virus(YFV)and chikungunya virus(CHIKV),so as to achieve the rapid detection of these three viruses.Methods The complete genome sequences of DENV(Ⅰ,Ⅱ,Ⅲ,Ⅳ),YFV and CHIKV were retrieved from Global Shared Database for comparative analysis,estimate its conservative region,specific primers and probes were designed,then a triplex real-time RT-PCR assay was developed.The specificity was evaluated by other viral nucleic acids.The sensitivity was evaluated by in vitro transcribed RNAs of DENV,YFV and CHIKV.The repeatability of the method was evaluated by independent repeated experiments with different concentrations of viral nucleic acids.DENV detection method was validated with dengue patient serum.YFV and CHIKV detection methods were validated with simulated positive samples.The sera from healthy people were used for negative validation.Results This method has no cross-reaction with other viral nucleic acids.The limit of detection(LOD)of DENV(Ⅰ、Ⅱ、Ⅲ、Ⅳ),YFV and CHIKV in vitro transcribed RNAs were less than 21.55 copies/PCR,21.25 copies/PCR,21.85 copies/PCR,22.75 copies/PCR,22 copies/PCR,45.65 copies/PCR.The standard deviation of Ct values of each concentration was less than 0.5 and the coefficient of variation was less than 3%.The positive rate of clinical and simulated positive samples was 100%,and the negative rate of healthy serum was 100%.Conclusions A triplex real-time fluorescent quantitative RT-PCR assay for DENV,YFV and CHIKV detection was established,and proved to be specific,sensitive and repetitive.

关 键 词：逆转录-聚合酶链反应 登革病毒 黄热病毒 基孔肯雅病毒 

分 类 号：R446.5[医药卫生—诊断学]