Consensus Amplification and Novel Multiplex Sequencing Method for S Segment Species Identification of 47 Viruses of the Orthobunyavirus, Phlebovirus, and Nairovirus Genera of the Family Bunyaviridae
Consensus Amplification and Novel Multiplex Sequencing Method for S Segment Species Identification of 47 Viruses of the Orthobunyavirus, Phlebovirus, and Nairovirus Genera of the Family Bunyaviridae
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DOI：
10.1128/jcm.00182-09
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发表时间：
2009-08-01
影响因子：
9.4
通讯作者：
Lanciotti, Robert S.
中科院分区：
文献类型：
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作者：
Lambert, Amy J.;Lanciotti, Robert S.
根据先前确定的和新获得的遗传数据，设计了一种逆转录-PCR（RT-PCR）检测方法，以靶向布尼亚病毒科47种病毒（包括29种节肢动物传播的人类病原体）的S基因组片段。通过将其应用于从感兴趣的布尼亚病毒的定量稀释液中提取的RNA，评价了所提出的测定法的分析灵敏度。此外，通过评价从选定的布尼亚病毒和其他代表性节肢动物传播病毒中提取的RNA，确定了该检测试剂盒的分析专属性，这些病毒分离自不同的宿主种属以及时间和地理来源。RT-PCR扩增后，从感兴趣的布尼亚病毒扩增的DNA进行了一种新的多重测序方法，以确认布尼亚病毒阳性，并提供初步的，物种水平的S片段鉴定。我们的目标是，该检测试剂盒将用作鉴别和表征紧急节肢动物传播的布尼亚病毒分离株（医用进口）以及与人类疾病无关的布尼亚病毒科相关病毒的工具。
A reverse transcription-PCR (RT-PCR) assay was designed, according to previously determined and newly derived genetic data, to target S genomic segments of 47 viruses, including 29 arthropod-borne human pathogens, of the family Bunyaviridae. The analytical sensitivity of the presented assay was evaluated through its application to RNAs extracted from quantitated dilutions of bunyaviruses of interest. Additionally, the assay's analytical specificity was determined through the evaluation of RNAs extracted from selected bunyaviruses and other representative arthropod-borne viruses isolated from a diverse group of host species and temporal and geographic origins. After RT-PCR amplification, DNAs amplified from bunyaviruses of interest were subjected to a novel multiplex sequencing method to confirm bunyavirus positivity and provide preliminary, species-level S segment identification. It is our goal that this assay will be used as a tool for identification and characterization of emergent arthropod-borne bunyavirus isolates of medical import as well as related viruses of the family Bunyaviridae that have not been associated with human illness.