Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
National Bio and Agro-Defense Facility, United States Department of Agriculture, Agricultural Research Service, Foreign Arthropod-Borne Animal Diseases Research Unit, Manhattan, KS, United States.
J Virol Methods. 2023 May;315:114693. doi: 10.1016/j.jviromet.2023.114693. Epub 2023 Feb 16.
Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT-qPCR genotyping (GT) assays were developed to differentiate between two RVFV wild-type strains (128B-15 and SA01-1322) and a vaccine strain (MP-12). The GT assay uses a one-step RT-qPCR mix, with two different RVFV strain-specific primers (either forward or reverse) with long or short G/C tags and a common primer (either forward or reverse) for each of the 3 genomic segments. The GT assay produces PCR amplicons with unique melting temperatures that are resolved in a post PCR melt curve analysis for strain identification. Furthermore, a strain specific RT-qPCR (SS-PCR) assay was developed to allow for specific detection of low titer RVFV strains in mixed RVFV samples. Our data shows that the GT assays are capable of differentiating L, M, and S segments of RVFV strains 128B-15 versus MP-12, and 128B-15 versus SA01-1322. The SS-PCR assay results revealed that it can specifically amplify and detect a low titer MP-12 strain in mixed RVFV samples. Overall, these two novel assays are useful as screening tools for determining reassortment of the segmented RVFV genome during co-infections, and could be adapted and applied for other segmented pathogens of interest.
裂谷热病毒（RVFV）是一种新兴的、由蚊子传播的、人畜共患病原体。实时 RT-qPCR 基因分型（GT）检测方法的开发是为了区分两种 RVFV 野生型毒株（128B-15 和 SA01-1322）和一种疫苗株（MP-12）。GT 检测方法使用一步法 RT-qPCR 混合物，带有两个不同的 RVFV 株特异性引物（正向或反向），带有长或短的 G/C 标记，以及每个 3 个基因组片段的通用引物（正向或反向）。GT 检测方法产生具有独特熔点的 PCR 扩增子，在 PCR 熔解曲线分析中进行分析以确定菌株身份。此外，还开发了一种针对 RVFV 株的 RT-qPCR（SS-PCR）检测方法，用于在混合 RVFV 样本中检测低滴度 RVFV 株。我们的数据表明，GT 检测方法能够区分 128B-15 株与 MP-12 株和 128B-15 株与 SA01-1322 株的 L、M 和 S 片段。SS-PCR 检测方法的结果表明，它可以特异性地扩增和检测混合 RVFV 样本中的低滴度 MP-12 株。总的来说，这两种新的检测方法可作为筛选工具，用于确定分段 RVFV 基因组在共同感染过程中的重组情况，并且可以适应和应用于其他感兴趣的分段病原体。
