Comparison of four methods to quantify equid herpesvirus 1 load by real-time polymerase chain reaction in nasal secretions of experimentally and naturally infected horses

Nicola Pusterla, Stephen B. Hussey, Samantha Mapes, Christian M. Leutenegger, John E Madigan, Gregory L. Ferraro, William D Wilson, D. Paul Lunn

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The objective of the current study was to compare the performance of 4 methods to quantify Equid herpesvirus 1 (EHV-1) by real-time polymerase chain reaction (PCR) in nasal secretions from experimentally and naturally infected horses. Nasal secretions were collected on the challenge day and daily thereafter for 13 days from 4 experimentally infected horses. Additional nasal swabs were collected from 30 horses with clinical signs consistent with natural EHV-1 infection. Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and equine glyceraldehyde-3-phosphate dehydrogenase, and DNA yield, and was expressed as EHV-1 glycoprotein B (gB) gene copies per million nucleated nasal cells, EHV-1 gB gene copies per entire swab, EHV-1 gB gene copies per 1 ml of purified DNA, and EHV-1 gB gene copies per 1 ng of template DNA. The study results showed that all 4 calculation methods yielded comparable results between experimentally and naturally infected horses, and that the different methods were significantly correlated with each other. Reporting of quantitative results for EHV-1 viral load in nasal swabs collected from infected horses constitutes an important advance in both the research and diagnostic fields, allowing one to determine the infectious risk of affected horses, disease stage, or response to antiviral therapy. However, protocols that normalize the PCR results against a preselected volume of DNA or nasal secretions are likely to be more prone to variations than protocols that calculate the load for the entire swab, incorporate a housekeeping gene, or use a constant amount of extracted DNA.

Original languageEnglish (US)
Pages (from-to)836-840
Number of pages5
JournalJournal of Veterinary Diagnostic Investigation
Volume21
Issue number6
StatePublished - Nov 1 2009

Keywords

  • Equid herpesvirus 1
  • Horses
  • Nasal secretions
  • Real-time polymerase chain reaction
  • Viral load quantitation methods

ASJC Scopus subject areas

  • veterinary(all)

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