Daleo Maris J, Allender Matthew C
Wildlife Epidemiology Laboratory, University of Illinois, Urbana, IL 61802, USA.
Wildlife Epidemiology Laboratory, University of Illinois, Urbana, IL 61802, USA; Chicago Zoological Society/Brookfield Zoo, Brookfield, IL 60513, USA.
J Virol Methods. 2025 Feb;332:115077. doi: 10.1016/j.jviromet.2024.115077. Epub 2024 Nov 22.
Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05-1.826 % across all concentrations, while inter-assay variation ranged from 0.031-4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (Trachemys scripta elegans) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.
许多野生动物保护工作聚焦于一种病原体的影响，但要使众多保护工作取得成功，研究人员需要了解可能包括多种同时存在的病原体的生态过程。我们开发了一种多重定量聚合酶链反应（qPCR）检测方法，用于检测东部箱龟（Terrapene carolina carolina）体内的四种病原体，包括蛙病毒3（FV3）、箱龟疱疹病毒1（TerHV1）、箱龟支原体（BTMyco）和箱龟腺病毒（TerAdv）。使用先前发表的检测方法并搭配四种不同的荧光团设计了TaqMan™引物探针。将多重反应的Cq值与单重反应的Cq值作图，结果显示TerHV1、TerAdv、FV3和BTMyco的斜率分别为0.967、1.00、0.980和0.973，且所有四种病原体的R值均为0.999。该检测方法与所有四种病原体靶标的批内变异高度一致，在所有浓度下变异范围为0.05 - 1.826%，而批间变异在所有浓度下所有四个靶标的范围为0.031 - 4.569%。使用先前从东部箱龟和红耳龟（Trachemys scripta elegans）收集的临床样本进行检测，其结果与单重检测方法相似。这种多重检测方法是一种有效、省时的诊断工具，可通过在单一反应中检测FV3、TerHV1、BTMyco和TerAdv来快速监测龟类病原体。一种经过验证且临床应用的多重检测方法将有助于为未来的龟类流行病学研究描绘更复杂的病原体图谱，从而更好地描述病原体动态及其对个体和种群健康的影响。
