Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.
Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.
复制标题
DOI：
10.1016/j.jmoldx.2015.06.008
复制
发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
Chu JJ
中科院分区：
文献类型：
--
作者：
Chen H;Parimelalagan M;Lai YL;Lee KS;Koay ES;Hapuarachchi HC;Ng LC;Ho PS;Chu JJ
基孔肯雅病毒（CHIKV）和登革热病毒（DENV）已成为两种最重要的虫媒病毒疾病的全球健康意义。临床表现、传播媒介、地理分布和季节相关性的相似往往导致基孔肯雅感染误诊为登革热病例，反之亦然。在这项研究中，我们开发了一种快速准确的实验室确认方法，可以同时检测，定量和区分DENV血清型1，2，3和4以及CHIKV。这种基于SYBR绿色I的一步多重实时RT-PCR检测方法对CHIKV和DENV具有高度灵敏度和特异性。使用PCR扩增子的解链温度分析对DENV进行血清分型并与CHIKV区分。对于DENV-1、DENV-2、DENV-3、DENV-4和CHIKV，测定的检测限分别为20、10、50、5和10个RNA拷贝/反应。我们的检测试剂与一组病毒没有交叉反应，包括其他黄病毒、甲病毒、流感病毒、人类肠道病毒和人类冠状病毒。在DENV和CHIKV阳性患者血清中评价了使用该测定进行临床诊断的可行性。因此，DENV-1、DENV-2、DENV-3、DENV-4和CHIKV的测定灵敏度分别为89.66%、96.67%、96.67%、94.12%和95.74%，特异性为100%。这些发现证实了我们的测定法用作临床样品中DENV和CHIKV的同时检测和血清分型的快速测试的潜力。
Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I–based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.
登录
查看更多内容
影响因子：
11.8
作者：
Tun MM;Thant KZ;Inoue S;Nabeshima T;Aoki K;Kyaw AK;Myint T;Tar T;Maung KT;Hayasaka D;Morita K
通讯作者：
Morita K
DOI：
10.4269/ajtmh.1980.29.638
发表时间：
1980-01-01
影响因子：
3.3
作者：
HALSTEAD, SB;PORTERFIELD, JS;OROURKE, EJ
通讯作者：
OROURKE, EJ
影响因子：
5.4
作者：
Midgley, Claire M.;Bajwa-Joseph, Martha;Screaton, Gavin R.
通讯作者：
Screaton, Gavin R.
影响因子：
1.7
作者：
Mohan, Alladi;Kiran, D H N;Kumar, D Prabath
通讯作者：
Kumar, D Prabath
影响因子：
5.4
作者：
Morrison, Thomas E.
通讯作者：
Morrison, Thomas E.