Usutu virus (USUV) forward primer complementing the Jost et al. primer set

We designed an additional forward primer, USUV_MM_for, to detect Usutu virus (USUV) in diagnostic samples that is compatible with the reverse primer as well as the probe of the primer pair published by Jöst et al. (2011). The combined primer pair produces a PCR product of 265 bp, making it more suitable for Sanger sequencing downstream. The primer pair was designed by aligning different USUV strains, including Africa 1 (KC754958), Africa 2 (AY453412), Africa 3.1 (MN122238), Africa 3.2 (MN122249), Europe 3 (MN122196) and WNV Lin1 (MW627239), selecting a region of the genome that is conserved for USUV but divergent for WNV. RT-qPCR analysis showed that the new forward primer (combined with the reverse Jost primer) amplified USUV but not WNV (lin 1 and lin 2) or RVFV (PCR report and gel image attached).我们设计了一款额外的正向引物USUV_MM_for，用于检测诊断样本中的乌图病毒（Usutu virus, USUV），该引物可与Jöst等人2011年发表的引物对的反向引物及探针兼容。该组合引物对可扩增得到长度为265 bp的PCR产物，更适用于下游桑格测序（Sanger sequencing）。本引物对的设计策略为：比对多株乌图病毒毒株，包括非洲1型（KC754958）、非洲2型（AY453412）、非洲3.1型（MN122238）、非洲3.2型（MN122249）、欧洲3型（MN122196）以及西尼罗病毒谱系1型（WNV Lin1, MW627239），筛选出乌图病毒基因组中保守、但西尼罗病毒（WNV）基因组中存在序列差异的区域。反转录实时定量聚合酶链式反应（RT-qPCR）分析结果显示，该新型正向引物与Jöst等使用的反向引物组合后，可特异性扩增乌图病毒，而非西尼罗病毒（谱系1型与谱系2型）或裂谷热病毒（RVFV, Rift Valley Fever Virus）（附带PCR检测报告与凝胶电泳图像）。