The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1.
Abstract: Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR-ELISA system. A competitive PCR-ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR-ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms.
Publication Date: 2002-12-31 PubMed ID: 12505639DOI: 10.1016/s0166-0934(02)00252-5Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research is about the development of a competitive PCR-ELISA system for the detection of Equine Herpesvirus-1 (EHV-1), a major issue for animal welfare and the economy. The research aims to overcome problems with current diagnostic approaches to EHV-1 infection by creating a fast and reliable detection system.
Understanding the Research
- The research was initiated due to the absence of standardised molecular techniques for diagnosing or confirming viral infection of EHV-1, an infection that has significant impact on animal welfare and the economy.
- The seminal aim was to develop a dependable, quantitative assay system capable of detecting EHV-1 and eliminating the occurrence of false negative results.
Developing the Assay System
- A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned, and then subjected to site-directed mutagenesis. This generated a control plasmid.
- The control plasmid can be amplified by identical primers to wild type EHV-1, but is capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR-ELISA system.
Results and Implications
- As a result, a competitive PCR-ELISA system was developed. This system can control for the presence of PCR inhibitors and is capable of detecting 63 genome equivalents of EHV-1.
- This new system was successfully used to demonstrate the presence of EHV-1 in infected equine tissue and cell culture material.
- Overall, the system can provide results within one working day, facilitating multiple sample analysis.
- Therefore, this competitive PCR-ELISA system offers the potential for rapid and sensitive detection of EHV-1. Amid the wider implications, it can potentially prevent the occurrence of abortion storms in stud farms by allowing early detection and prompt intervention of EHV-1 infections.
Cite This Article
APA
Daly P, Doyle S.
(2002).
The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1.
J Virol Methods, 107(2), 237-244.
https://doi.org/10.1016/s0166-0934(02)00252-5 Publication
Researcher Affiliations
- National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth Co., Kildare, Ireland.
MeSH Terms
- Abortion, Veterinary / diagnosis
- Abortion, Veterinary / virology
- Animals
- Enzyme-Linked Immunosorbent Assay
- Female
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Male
- Oligonucleotide Probes
- Plasmids
- Polymerase Chain Reaction / methods
- Pregnancy
- Sensitivity and Specificity
- Time Factors
- Viral Envelope Proteins / genetics
Citations
This article has been cited 4 times.- Titov I, Rutschke N, Kraft FA, Köpke M, Nebling E, Gerken M. Detection of fluorescence-labeled DNA with in-plane organic optoelectronic devices. Biomed Opt Express 2022 Dec 1;13(12):6300-6316.
- Yildirim Y, Yilmaz V, Kirmizigul AH. Equine herpes virus type 1 (EHV-1) and 4 (EHV-4) infections in horses and donkeys in northeastern Turkey. Iran J Vet Res 2015 Fall;16(4):341-4.
- Cleri DJ, Ricketti AJ, Porwancher RB, Ramos-Bonner LS, Vernaleo JR. Viral hemorrhagic fevers: current status of endemic disease and strategies for control. Infect Dis Clin North Am 2006 Jun;20(2):359-93, x.
- Mousavi-Rad SMA, Zare Karizi S, Sedighian H, Mirhosseini SA, Esmaeili Gouvarchin Ghaleh H, Amani J. Design and comparison of PCR-ELISA reaction with other available hybridization methods to identify types 11, 16, and 18 of the human papillomavirus. Iran J Microbiol 2025 Jun;17(3):488-502.
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