Herpesviruses are associated with disease in many chelonian species, resulting in pathology such as respiratory tract infection, stomatitis, conjunctivitis, hepatitis, and papillomatosis. Herpesvirus-associated fibropapillomas cause significant morbidity and mortality in marine turtles, and have been identified in an eastern box turtle (Terrapene carolina carolina) infected with Terrapene herpesvirus 2 (TerHV2). Further investigation is necessary to understand the impact of carcinogenic herpesviruses on chelonian health; however, reliable and specific methods for detection and quantitation of herpesviral load are lacking. The purpose of this study was to develop and validate a quantitative PCR assay for detection of TerHV2 in box turtles. TaqMan primer-probes were developed targeting the DNA polymerase gene. Inter- and intra-assay variability, linear range of detection, limit of detection, and specificity were assessed. The assay was highly specific for TerHV2, failing to amplify seven closely-related chelonian herpesviruses. It performed with high efficiency (slope = -3.52, R² = 1, efficiency = 92.29 %), low intra-assay variability and low inter-assay variability (coefficient of variation ≤ 1.25 % at all standard dilutions). Reaction efficiency was not impacted in the presence of box turtle DNA from combined oral/cloacal swabs or whole blood. This qPCR assay has a linear range of detection from 10⁷ to 10¹ viral copies per reaction and provides a valuable tool in the surveillance and characterization of TerHV2 in box turtles.

疱疹病毒与许多龟类的疾病有关，导致呼吸道感染、口腔炎、结膜炎、肝炎和乳头状瘤病等病理。疱疹病毒相关的纤维乳头状瘤在海龟中引起显着的发病率和死亡率，并已在感染了Terrapene的东部箱龟（Terrapene carolina carolina）中发现疱疹病毒 2 (TerHV2)。需要进一步调查以了解致癌疱疹病毒对龟类健康的影响；然而，缺乏用于检测和定量疱疹病毒载量的可靠且特定的方法。本研究的目的是开发和验证用于检测箱龟 TerHV2 的定量 PCR 检测方法。TaqMan 引物探针是针对 DNA 聚合酶基因开发的。评估了测定间和测定内的变异性、检测的线性范围、检测限和特异性。该测定对 TerHV2 具有高度特异性，未能扩增七种密切相关的龟类疱疹病毒。它以高效率执行（斜率 = -3.52，R ² = 1，效率 = 92.29 %），测定内变异性和测定间变异性低（变异系数≤ 1.25 % 在所有标准稀释度下）。在存在来自组合的口腔/泄殖腔拭子或全血的箱龟 DNA 的情况下，反应效率不会受到影响。该 qPCR 检测具有每次反应10 ⁷到 10 ¹病毒拷贝的线性检测范围，并为箱龟中 TerHV2 的监测和表征提供了有价值的工具。