Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5.
Abstract: Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.
Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.
Publication Date: 2022-09-08 PubMed ID: 36087793DOI: 10.1016/j.jviromet.2022.114615Google Scholar: Lookup
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Summary
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The research article presents a study on the development of a multiplex real-time PCR (rtPCR) for detecting and differentiating Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) in horses. The assay proves to be an efficient and swift tool for identifying these viruses, vital for conducting further studies on their clinical significance.
Introduction to the Equid gammaherpesvirus
- Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are prevalent in horses worldwide.
- Their complete pathogenic potential is not yet clear, though they are suggested to be a factor involved in equine diseases such as equine multinodular pulmonary fibrosis.
- Identifying and understanding these viruses is essential for exploring their role in the horse diseases in more depth.
Development of the Real-time PCR (rtPCR)
- The researchers designed a multiplex real-time PCR (rtPCR) to detect the DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5), indicative of the presence of the viruses.
- The rtPCR’s analytical specificity was determined by testing DNA of other herpesviruses.
- This testing was done using SYBR Green rtPCR and melting curve analysis, and through Sanger sequencing of positive field samples.
Evaluation of the rtPCR
- The rtPCR’s analytical sensitivity was evaluated using standard curves generated from serial plasmid dilutions containing the target gene for each virus.
- Specific detection of the viruses was confirmed through analysis of the melting curves and BLAST analysis of the sequences.
- The lower limit of detection, which refers to the smallest quantity of a substance that can be reliably measured by the assay, was found to be 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively.
Comparison to Other Assays
- When compared with singleplex assays (tests that detect a single target in one reaction), the new multiplex rtPCR showed minimal differences for a selection of positive field samples.
- The minimal differences suggest a comparable performance level between the new multiplex rtPCR and existing singleplex assays.
Conclusion
- The developed multiplex rtPCR offers sensitive and specific detection of EHV-2 and EHV-5.
- This new assay provides a convenient and rapid method for further investigations on the clinical importance of these viruses in equine diseases.
Cite This Article
APA
Fürer F, Fraefel C, Lechmann J.
(2022).
Multiplex real-time PCR for the detection and differentiation of equid gammaherpesvirus 2 and 5.
J Virol Methods, 310, 114615.
https://doi.org/10.1016/j.jviromet.2022.114615 Publication
Researcher Affiliations
- Institute of Virology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland.
- Institute of Virology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland.
- Institute of Virology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland. Electronic address: julia.lechmann@uzh.ch.
MeSH Terms
- Horses
- Animals
- Real-Time Polymerase Chain Reaction / methods
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / veterinary
- Horse Diseases / diagnosis
- DNA, Viral / genetics
- Herpesviridae / genetics
- Herpesvirus 1, Equid / genetics
- Herpesvirus 4, Equid / genetics
Conflict of Interest Statement
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Citations
This article has been cited 3 times.- Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V. Quadruplex Real-Time TaqMan(®) RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes. Viruses 2023 Jul 26;15(8).
- Zhang B, Qiu Y, Shi C, Zhang J. Development of Multiple Real-Time Fluorescent Quantitative PCR for Vibrio Pathogen Detection in Aquaculture. Vet Sci 2025 Apr 2;12(4).
- Li L, Li S, Ma H, Akhtar MF, Tan Y, Wang T, Liu W, Khan A, Khan MZ, Wang C. An Overview of Infectious and Non-Infectious Causes of Pregnancy Losses in Equine. Animals (Basel) 2024 Jul 2;14(13).
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