Herpesviruses are associated with disease in many penguin species. Herpesvirus-associated lesions can cause significant morbidity and mortality in penguins and have been identified in African penguins ( Spheniscus demersus), Humboldt penguins ( Spheniscus humboldti), and a little blue penguin ( Eudyptula minor) infected with spheniscid alphaherpesvirus 1 (SpAHV1). Further investigation is necessary to understand the impact of herpesviruses on penguin health, but there are no rapid, sensitive, and specific methods for detecting and quantifying herpesviral load. We therefore developed a quantitative real-time PCR (qPCR) assay for the detection of SpAHV1 in penguins. TaqMan primer-probes targeting the DNA polymerase gene were designed using a commercial software program. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed. We used our assay to analyze previously collected field samples from Punta San Juan, Peru, in which conventional consensus PCR had detected one SpAHV1-positive penguin sample. Our qPCR assay was highly specific for SpAHV1. It had a dynamic range of 107–101 target copies per reaction and performed with high efficiency and low intra- and inter-assay variability. Reaction efficiency was not impacted by penguin DNA from SpAHV1-negative tracheal swabs. We detected an additional field sample as positive with our newly developed qPCR assay, and although this likely represents detection of another infected penguin, the true disease status of this population is currently uncharacterized given that no gold-standard test exists for SpAHV1. Our qPCR assay may provide a valuable tool in the surveillance and characterization of SpAHV1 in penguins.

中文翻译：

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用于检测企鹅中 α 疱疹病毒 1 型的实时定量 PCR 检测的开发和分析特性

疱疹病毒与许多企鹅物种的疾病有关。疱疹病毒相关病变可导致企鹅显着发病和死亡，并已在感染 spheniscid α疱疹病毒 1 (SpAHV1) 的非洲企鹅 (Spheniscus demersus)、洪堡企鹅 (Spheniscus humboldti) 和小蓝企鹅 (Eudyptulaminor) 中发现。为了了解疱疹病毒对企鹅健康的影响，有必要进行进一步的研究，但目前还没有快速、灵敏和具体的方法来检测和量化疱疹病毒载量。因此，我们开发了一种定量实时 PCR (qPCR) 检测方法来检测企鹅中的 SpAHV1。使用商业软件程序设计针对 DNA 聚合酶基因的 TaqMan 引物探针。评估了测定间和测定内的变异性、动态范围、检测限和分析特异性。我们使用我们的检测方法分析了之前从秘鲁蓬塔圣胡安收集的现场样本，其中常规 PCR 检测到了一份 SpAHV1 阳性企鹅样本。我们的 qPCR 检测对 SpAHV1 具有高度特异性。它的动态范围为 107–101每个反应的目标拷贝数高，且检测内和检测间变异性低。SpAHV1 阴性气管拭子中的企鹅 DNA 不会影响反应效率。我们通过新开发的 qPCR 检测方法检测到了额外的现场样本呈阳性，尽管这可能代表检测到了另一只受感染的企鹅，但鉴于不存在 SpAHV1 的金标准测试，因此该种群的真实​​疾病状况目前尚未确定。我们的 qPCR 检测可能为企鹅 SpAHV1 的监测和表征提供有价值的工具。