中国人兽共患病学报2024，Vol.40Issue(6)：537-543,7.DOI:10.3969/j.issn.1002-2694.2024.00.080
含内参的登革和寨卡病毒三重RT-qPCR检测方法的建立与评估
Development and evaluation of a triplex RT-qPCR assay with internal references for detection of the Dengue and Zika viruses
摘要
Abstract
A triplex RT-qPCR assay with human genes as internal references was established for detection of the Dengue and Zika viruses(DENV and ZIKV,respectively).The conserved regions of the four serotypes of DENV,along with the NS1 gene of ZIKV and the human β-actin gene,which is stably ex-pressed in various human tissues,were targeted by three sets of specific primers and probes.Standard plasmids for four se-rotypes of DENV,ZIKV,and β-actin were constructed as pos-itive controls.Optimal reaction conditions were determined through an L9(34)orthogonal experiment.The specificity,sensitivity,and coverage of the assay were verified and evalua-ted clinically,and the consistency was evaluated against a com-mercial kit for detection of DENV.The triplex RT-qPCR assay established exhibited no non-specific cross reactions with 12 similar arboviruses.The detection sensitivity for DENV and ZIKV were 2.99 and 2.18 copies/μL,respectively,and the intra-group and inter-group repeatability coefficients of variation were within 1.5％.As compared to the commercial kit,the proposed assay obtained positive results for 13 epidemic strains of DENV.Bland-Altman consistency analysis confirmed that the consistency of the detection results of clinical positive samples between the commercial kit and the proposed assay was 92.59％.The highly specific and sensitive triplex RT-qPCR assay with internal references is an effective tool for early and rapid differential identification of DENV and ZIKV.关键词
三重RT-qPCR/登革病毒/寨卡病毒/内参Key words
triplex RT-qPCR/Dengue virus/Zika virus/internal reference分类
医药卫生引用本文复制引用
曹孟涛,胡潇予,杨微,李春缘,徐晓立,任瑞文,蒋红霞..含内参的登革和寨卡病毒三重RT-qPCR检测方法的建立与评估[J].中国人兽共患病学报,2024,40(6):537-543,7.基金项目
全军实验动物专项科研课题(No.SYDW[2018]04)和广东省省级科技计划项目(No.2016A020219006)联合资助 Supported by the Experimental Animal Special Research Subject of the Whole Army(No.SYDW[2018]04)and the Provincial Science and Technology Plan Project of Guangdong(No.2016 A020219006) （No.SYDW[2018]04）
