Development and validation of a quantitative PCR assay for detection of Emydoidea herpesvirus 1 in free-ranging Blanding's turtles (Emydoidea blandingii)

Dana M. Lindemann, Matthew C Allender, Dan Thompson, Laura Adamovicz, Elena Dzhaman

Research output: Contribution to journalArticle

Abstract

Blanding's turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding's turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = −3.2; R 2 = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation <2% at all dilutions). However, EBHV1 primer-2 displayed less variation and was selected to test clinical samples and five closely related herpesvirus control samples. Results indicate that this assay is specific for EBHV1, has a linear range of detection from 10 8 to 10 1 viral copies per reaction, and can categorically detect as few as 1 viral copy per reaction. This qPCR assay provides a valuable diagnostic tool for future characterization of EBHV1 epidemiology.

Original languageEnglish (US)
Pages (from-to)40-45
Number of pages6
JournalJournal of Virological Methods
Volume254
DOIs
StatePublished - Apr 2018

Fingerprint

Turtles
Herpesviridae
Polymerase Chain Reaction
Fresh Water
Endangered Species
Mortality
DNA-Directed DNA Polymerase
Sequence Homology
Natural History
Population
Disease Outbreaks
Communicable Diseases
Ecosystem
Real-Time Polymerase Chain Reaction
Epidemiology
Demography
Morbidity

Keywords

  • Blanding's turtle
  • Chelonian
  • Emydoidea blandingii
  • Epidemiology
  • Herpesvirus
  • qPCR

ASJC Scopus subject areas

  • Virology

Cite this

Development and validation of a quantitative PCR assay for detection of Emydoidea herpesvirus 1 in free-ranging Blanding's turtles (Emydoidea blandingii). / Lindemann, Dana M.; Allender, Matthew C; Thompson, Dan; Adamovicz, Laura; Dzhaman, Elena.

In: Journal of Virological Methods, Vol. 254, 04.2018, p. 40-45.

Research output: Contribution to journalArticle

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abstract = "Blanding's turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding's turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = −3.2; R 2 = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation <2{\%} at all dilutions). However, EBHV1 primer-2 displayed less variation and was selected to test clinical samples and five closely related herpesvirus control samples. Results indicate that this assay is specific for EBHV1, has a linear range of detection from 10 8 to 10 1 viral copies per reaction, and can categorically detect as few as 1 viral copy per reaction. This qPCR assay provides a valuable diagnostic tool for future characterization of EBHV1 epidemiology.",
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