Development of a universal RT-PCR for amplifying and sequencing the leader and capsid-coding region of foot-and-mouth disease virus
Development of a universal RT-PCR for amplifying and sequencing the leader and capsid-coding region of foot-and-mouth disease virus
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DOI：
10.1016/j.jviromet.2013.01.009
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发表时间：
2013-04-01
影响因子：
3.1
通讯作者：
McIntosh, Michael T.
中科院分区：
文献类型：
--
作者：
Xu, Lizhe;Hurtle, William;McIntosh, Michael T.
口蹄疫（Foot-and-mouth disease，FMD）是一种传染性极强的偶蹄类动物病毒性疾病，对世界各地的畜牧业造成严重的危害。致病因子FMD病毒（FMDV）的关键抗原决定簇存在于病毒衣壳的表面暴露的蛋白质内。因此，编码衣壳（P1）的序列的表征对于追踪FMD的出现或传播以及新疫苗的选择和开发是重要的。由于FMDV不同毒株之间的高血清型间变异性，产生该区域序列的可靠方法具有挑战性。本研究描述了一种新的、稳健的和通用的RT-PCR方法的开发和优化，该方法可用于扩增和测序包含FMDV基因组的前导蛋白酶（L）和衣壳蛋白编码部分（P1）的3 kb片段。这种新的RT-PCR方法在两个实验室进行了评估，使用从不同国家收集的134份临床样本中提取的RNA，代表了7种FMDV血清型中每一种血清型的一系列拓扑型和谱系。序列分析有助于重复设计适合于这些RT-PCR片段的常规测序的引物。利用该方法对从现场采集的49株口蹄疫病毒进行了序列分析。这种方法提供了一个强大的工具，可用于快速抗原特性的FMDV和系统发育分析，并已列入实验室响应程序作为一个援助疫苗匹配或选择在口蹄疫爆发的情况下的效用。由爱思唯尔公司出版
Foot-and-mouth disease (FMD) is a highly infectious viral disease of cloven-hoofed animals with debilitating and devastating consequences for livestock industries throughout the world. Key antigenic determinants of the causative agent, FMD virus (FMDV), reside within the surface-exposed proteins of the viral capsid. Therefore, characterization of the sequence that encodes the capsid (P1) is important for tracking the emergence or spread of FMD and for selection and development of new vaccines. Reliable methods to generate sequence for this region are challenging due to the high inter-serotypic variability between different strains of FMDV. This study describes the development and optimization of a novel, robust and universal RT-PCR method that may be used to amplify and sequence a 3 kilobase (kb) fragment encompassing the leader proteinase (L) and capsid-coding portions (P1) of the FMDV genome. This new RT-PCR method was evaluated in two laboratories using RNA extracted from 134 clinical samples collected from different countries and representing a range of topotypes and lineages within each of the seven FMDV serotypes. Sequence analysis assisted in the reiterative design of primers that are suitable for routine sequencing of these RT-PCR fragments. Using this method, sequence analysis was undertaken for 49 FMD viruses collected from outbreaks in the field. This approach provides a robust tool that can be used for rapid antigenic characterization of FMDV and phylogenetic analyses and has utility for inclusion in laboratory response programs as an aid to vaccine matching or selection in the event of FMD outbreaks. Published by Elsevier B.V.