Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA
Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA
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DOI：
10.1016/j.jviromet.2013.07.050
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发表时间：
2013-12-01
影响因子：
3.1
通讯作者：
Martina, Byron E. E.
中科院分区：
文献类型：
--
作者：
Lim, Stephanie M.;Koraka, Penelope;Martina, Byron E. E.
研究西尼罗病毒（WNV）在体外不同细胞类型和动物模型组织中的嗜性和复制动力学对于了解其发病机制具有重要意义。由于检测到负链病毒RNA是单链正义RNA病毒活跃复制的更可靠指标，因此重新评估了目前用于检测WNV正链和负链RNA的qRT-PCR检测方法的特异性。结果表明，在使用未修饰的引物和几种逆转录酶的测定中，在逆转录步骤期间产生了自引发和假引发的cDNA。因此，开发了使用热稳定性rTth与标记引物组合的qRT-PCR测定，其通过避免自引发和假引发事件而大大提高了链特异性。然后使用BV-2小胶质细胞以及C57/BL 6小鼠在体外解决了该测定的可靠性。可以在体外和体内跟踪正链和负链RNA合成的动力学;然而，需要优化测定的灵敏度，以便在感染的非常早期阶段检测和定量负链RNA合成。总体而言，本研究中开发的链特异性qRT-PCR检测是定量WNV RNA、重新评估病毒复制和研究WNV发病机制背景下WNV嗜性的有效工具。(C)2013爱思唯尔有限公司版权所有。
Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types in vitro and in tissues in animal models is important for understanding its pathogenesis. As detection of the negative strand viral RNA is a more reliable indicator of active replication for single-stranded positive-sense RNA viruses, the specificity of qRT-PCR assays currently used for the detection of WNV positive and negative strand RNA was reassessed. It was shown that self-and falsely-primed cDNA was generated during the reverse transcription step in an assay employing unmodified primers and several reverse transcriptases. As a result, a qRT-PCR assay using the thermostable rTth in combination with tagged primers was developed, which greatly improved strand specificity by circumventing the events of self- and false-priming. The reliability of the assay was then addressed in vitro using BV-2 microglia cells as well as In C57/BL6 mice. It was possible to follow the kinetics of positive and negative-strand RNA synthesis both in vitro and in vivo; however, the sensitivity of the assay will need to be optimized in order to detect and quantify negative-strand RNA synthesis in the very early stages of infection. Overall, the strand-specific qRT-PCR assay developed in this study is an effective tool to quantify WNV RNA, reassess viral replication, and study tropism of WNV in the context of WNV pathogenesis. (C) 2013 Elsevier B.V. All rights reserved.