Development and Evaluation of a Simple Assay for Marburg Virus Detection Using a Reverse Transcription-Loop-Mediated Isothermal Amplification Method
Development and Evaluation of a Simple Assay for Marburg Virus Detection Using a Reverse Transcription-Loop-Mediated Isothermal Amplification Method
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DOI：
10.1128/jcm.01224-09
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发表时间：
2010-07-01
影响因子：
9.4
通讯作者：
Yasuda, Jiro
中科院分区：
文献类型：
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作者：
Kurosaki, Yohei;Grolla, Allen;Yasuda, Jiro
马尔堡病毒(Marburg Virus，Marv)在人类中引起严重的出血热，死亡率很高。需要对病毒进行快速和准确的识别，以便适当地提供感染控制和暴发管理。在这里，我们建立并评价了一步反转录-环介导的等温扩增(RT-LAMP)方法，用于快速、简单地检测Marv。通过结合两套针对Musoke和Ravn基因谱系的引物，多重RT-LAMP检测到了这两个谱系的Marv毒株，并且没有观察到与其他出血热病毒(埃博拉病毒和拉萨病毒)的交叉反应。通过实时监测反应混合物的浊度，该方法可以在40分钟内检测到每管10(2)个拷贝的病毒RNA。使用从2005年安哥拉马尔堡出血热暴发期间收集的临床标本中提取的病毒RNA对该检测进行了进一步评估，对含有组织培养感染量/毫升10(4)%以上的MARV的样品产生了阳性结果，与现场实验室进行的逆转录-聚合酶链式反应检测结果的一致性为78%(18个样品中有14个为阳性)。琼脂糖凝胶电泳法和肉眼鉴定结果表明，本研究建立的RT-LAMP方法是一种有效的Marv分子检测工具。此外，它似乎适合用于现场诊断或在马立克氏病毒流行地区的实验室。
Marburg virus (MARV) causes a severe hemorrhagic fever in humans with a high mortality rate. The rapid and accurate identification of the virus is required to appropriately provide infection control and outbreak management. Here, we developed and evaluated a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and simple detection of MARV. By combining two sets of primers specific for the Musoke and Ravn genetic lineages, a multiple RT-LAMP assay detected MARV strains of both lineages, and no cross-reactivity with other hemorrhagic fever viruses (Ebola virus and Lassa virus) was observed. The assay could detect 10(2) copies of the viral RNA per tube within 40 min by real-time monitoring of the turbidities of the reaction mixtures. The assay was further evaluated using viral RNA extracted from clinical specimens collected in the 2005 Marburg hemorrhagic fever outbreak in Angola and yielded positive results for samples containing MARV at greater than 10(4) 50% tissue culture infective doses/ml, exhibiting 78% (14 of 18 samples positive) consistency with the results of a reverse transcription-PCR assay carried out in the field laboratory. The results obtained by both agarose gel electrophoresis and naked-eye judgment indicated that the RT-LAMP assay developed in this study is an effective tool for the molecular detection of MARV. Furthermore, it seems suitable for use for field diagnostics or in laboratories in areas where MARV is endemic.