Development and analytical validation of a novel quantitative PCR assay for the detection of Trachemys herpesvirus 1

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 10⁷ to 1.0 × 10¹ copies per reaction with an R² of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 10¹ copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.