Development and Validation of a Novel Quantitative PCR Assay for Detection of Emydid Herpesvirus 1

Herpesviruses are among the most widely understood and detrimental pathogens affecting chelonian populations worldwide. Mortality is not an uncommon consequence of these viruses, as has been documented with emydid herpesvirus 1 (EmyHV-1) infection in an eastern river cooter (Pseudemys concinna concinna), three western pond turtles (Actinemys marmorata), and a northern map turtle (Graptemys geographica). Due to the lack of targeted studies and available diagnostic tests for detection, the true occurrence and prevalence of EmyHV-1 is largely unknown. To aid in the epidemiological characterization of this virus, this study aimed to develop a sensitive and specific quantitative PCR assay for the detection of EmyHV-1 DNA in chelonian samples. A TaqMan-MGB FAM-dye labeled primer-probe set was designed to target a 56-base-pair segment of the EmyHV-1 DNA polymerase gene. This assay was specific for EmyHV-1 DNA, having failed to amplify eight other chelonian herpesviruses and demonstrating a dynamic range of 1.0×107 to 1.0×101 copies per reaction, a slope of –3.43, and a coefficient of determination (R2) of 0.999. Assay efficiency was unimpeded in clinical sample matrices of combined cloacal-oral swab DNA samples. The limit of detection for this assay was 101 copies per reaction, compared to 106 for a consensus PCR assay. Therefore, the qPCR assay outlined in this study demonstrates enhanced utility in the epidemiological characterization of EmyHV-1 in chelonians.