title: Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina).
abstract: Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04 × 10(7) to 1.04 × 10(1) viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease.
authors.affiliation: Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States. Electronic address: kane8@illinois.edu.
authors.forename: Lauren P
authors.initials: LP
authors.lastname: Kane
authors.affiliation: Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States.
authors.forename: David
authors.initials: D
authors.lastname: Bunick
authors.affiliation: Virology Department, College of Veterinary Medicine, Suez Canal University Faculty of Veterinary Medicine, Ismailia, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Abd-Eldaim
authors.affiliation: Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States.
authors.forename: Elena
authors.initials: E
authors.lastname: Dzhaman
authors.affiliation: Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, United States.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Allender
mesh_terms: D000818:Animals; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014426:Turtles / Q000821:virology*
chemical_list: D004259:DNA-Directed DNA Polymerase
keywords: Eastern box turtle; Epidemiology; Herpesvirus; Terrapene carolina; qPCR