登革病毒的Y型引物四重荧光定量RT-PCR方法的建立与评价  

Establishment and evaluation of Y-type primer quadruple fluorescence quantitative RT-PCR method for dengue virus

作　　者：史蕾[1] 徐云庆[1] 徐媛[1] Shi Lei;Xu Yunqing;Xu Yuan(Shenzhen International Travel Health Care Center,Shenzhen 518045,China)

机构地区：[1]深圳国际旅行卫生保健中心,深圳518045

出　　处：《中华实验和临床病毒学杂志》2022年第1期111-115,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划(2016YFF0203203)。

摘　　要：目的利用Y型引物和荧光定量RT-PCR技术建立一种用于登革病毒(dengus virus,DENV)快速分型的四重荧光定量RT-PCR检测方法。方法用DENV四种分型体外转录RNA对该方法的灵敏度、特异性进行评价及用临床样本进行验证。结果DENVⅠ型、Ⅱ型、Ⅲ型和Ⅳ型最低检出限分别为5.01反应/PCR、6.16拷贝/反应、6.35拷贝/反应、6.39拷贝/反应,与单重实时荧光定量RT-PCR比,最低检测限无明显变化;且与其他病毒无交叉反应,临床标本的阳性一致率和阴性一致率均为100%。结论本研究建立的Y型引物四重荧光定量RT-PCR方法可有效避免不同DENV分型间的交叉反应,同时具有良好的灵敏度和特异性,可用于相关临床标本的DENV快速分型检测。Objective This study aimed to establish a fourfold fluorescent quantitative RT-PCR detection method for rapid typing of Dengue virus(DENV)using Y-type primers and fluorescent quantitative RT-PCR technology.Methods Four types of DENV were used to transcribe RNA in vitro to evaluate the sensitivity and specificity of the method and to verify it with clinical samples.Results The minimum detection limits of DENV type I,II,III and IV were 5.01 copies/PCR,6.16 copies/PCR,6.35 copies/PCR,6.39 copies/PCR,respectively and had no significant difference compared with single-plex real-time fluorescent quantitative RT-PCR.There was also no crossover with other viruses.The positive agreement rate and negative agreement rate of clinical specimens were both 100%.Conclusions The Y-type primer quadruple fluorescence quantitative RT-PCR method established in this study could effectively avoid cross-reactions between different DENV types,and has good sensitivity and specificity,and can be used for rapid typing of DENV in relevant clinical specimens with high detection efficiency and low costs.

关 键 词：Y型引物 四重荧光定量RT-PCR 登革病毒 基因分型 

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