Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians

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 R² 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.