Development and validation of a quantitative PCR assay for detection of Terrapene herpesvirus 2 in eastern box turtles (Terrapene carolina carolina)

Aubrey I. Engel, Laura Adamovicz, James F.X. Wellehan, Matthew C. Allender

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article number113968
JournalJournal of Virological Methods
Volume286
DOIs
StatePublished - Dec 2020

Keywords

  • Chelonian
  • Eastern box turtle
  • Epidemiology
  • Terrapene carolina
  • Terrapene herpesvirus 2
  • qPCR

ASJC Scopus subject areas

  • Virology

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