Institute of Virology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland.
Institute of Virology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland.
J Virol Methods. 2022 Dec;310:114615. doi: 10.1016/j.jviromet.2022.114615. Epub 2022 Sep 8.
Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.
马γ疱疹病毒 2 型（EHV-2）和 5 型（EHV-5）广泛分布于马属动物中。尽管它们的致病潜力尚未完全了解，但它们似乎在马多发性肺纤维化等疾病模式中发挥作用。在本研究中，设计了一种多重实时 PCR（rtPCR）来检测糖蛋白 H（EHV-2）和 E11 基因（EHV-5）的 DNA。通过 SYBR Green rtPCR 和熔解曲线分析以及阳性现场样本的 Sanger 测序来测试其他疱疹病毒的 DNA，确定了分析特异性。通过包含相应靶基因的系列质粒稀释液的标准曲线生成来评估分析灵敏度。熔解曲线和序列的 BLAST 分析表明病毒的特异性检测。单重 rtPCR 的检测下限分别为 EHV-2 和 EHV-5 的每个反应 40 和 29 个 DNA 拷贝。对一组阳性现场样本的 Ct 值进行比较表明，单重和多重检测之间仅存在微小差异。本文描述的多重 rtPCR 方案可灵敏且特异性地检测 EHV-2 和 EHV-5。它代表了一种方便、快速的工具，可用于未来的研究，更详细地研究 EHV-2 和 EHV-5 的临床相关性。
