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利用多重扩增 PCR 技术高效、及时地生成裂谷热病毒序列数据进行基因组监测。

Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance.

机构信息

International Livestock Research Institute (ILRI), Nairobi P.O. Box 30709, Kenya.

South African National Bioinformatics Institute (SANBI), University of the Western Cape, Cape Town 7535, South Africa.

出版信息

Viruses. 2023 Feb 9;15(2):477. doi: 10.3390/v15020477.

Abstract

Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus in the family . It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses.

摘要

裂谷热(RVF)是一种在非洲流行的发热性虫媒病,并且继续在新的地区传播。它是一种主要由异常降雨模式引发的对气候敏感的疾病。该疾病与人类和牲畜的高死亡率和高发病率有关。RVF 是由裂谷热病毒(RVFV)引起的,属于 科 属。它是一种具有三个基因组片段的三分体 RNA 病毒:小(S)、中(M)和大(L)。病原体基因组测序正在成为一种常规程序,也是了解包括病毒在内的传染病原体进化动态的有力工具。受基于扩增子测序在严重急性呼吸综合征冠状病毒 2 型(SARS-CoV-2)、埃博拉、寨卡和西尼罗河病毒中的应用的启发,我们报告了一种裂谷热病毒样品制备方法,该方法基于扩增子多重聚合酶链反应(amPCR)进行模板富集,并减少背景宿主污染。该技术可以快速实施,以近乎实时的方式在暴发期间对裂谷热病毒进行特征分析和基因分型。为了实现这一目标,我们设计了 74 个多重引物组,涵盖了整个 RVFV 基因组,以专门扩增来自动物组织的临床样本中的 RVFV 核酸。使用这种方法,我们证明即使在含有相对低病毒载量的样本中也能实现 RVFV 全基因组覆盖。我们报告了第一个用于可复制其他分段病毒的三分体病毒的生成多重引物组的引物方案方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e34d/9961268/927de6daa619/viruses-15-00477-g001.jpg

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