Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens.
Abstract: Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duplex PCR was developed for the detection and differentiation of both ERAV and ERBV. The method was evaluated for its ability to detect viral RNA in cell culture supernatants and nasal swabs, and lung and urine spiked with known quantities of virus. The assay demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation (CV%) ranging from 1% to 7.4% and from 1.2% to 12% for intra- and inter-assay variability respectively. The assay detected ERBV in 14 of 86 nasal swabs collected from horses with respiratory disease. The real-time duplex PCR is a useful new diagnostic method for the rapid detection and differentiation of ERAV and ERBV.
Publication Date: 2008-12-02 PubMed ID: 19013197DOI: 10.1016/j.jviromet.2008.10.009Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article is about the development of a new diagnostic method known as real-time duplex PCR for detecting Equine rhinitis A and B viruses (ERAV and ERBV) in horses.
Objective of the Study
The main objective of the study was to create a reliable method for detecting and differentiating between Equine rhinitis A and B viruses, two respiratory viruses in horses. Current testing methods aren’t effective as these viruses show limited growth and don’t cause detectable changes in cell cultures.
The New Diagnostic Method
In this study, researchers developed a novel diagnostic method called a real-time duplex PCR (Polymerase Chain Reaction). Some of its key features include:
- Real-time analysis: This Polymerase Chain Reaction method allows ongoing observation of the process, providing real-time results.
- Duplex nature: It simultaneously tests for both ERAV and ERBV, eliminating the need for individual testing.
Evaluation of the Method
Researchers evaluated the real-time duplex PCR method’s effectiveness using the following criteria:
- Cell culture supernatants and nasal swabs, lung, and urine samples spiked with known amounts of the virus were tested.
- The method showed high specificity and sensitivity, and good reproducibility, making it a reliable testing mechanism.
- Variability within the same assay and between different assays was also assessed, showing a coefficient of variation ranging from 1% to 7.4% for intra-assay variability and 1.2% to 12% for inter-assay variability.
Results
The duplex PCR was used on 86 nasal swabs collected from horses with respiratory disease:
- ERBV was detected in 14 samples, confirming its suitability for detecting these viruses in clinical specimens.
Conclusion
The real-time duplex PCR is a valuable tool for rapidly detecting and differentiating between ERAV and ERBV in horses. This new diagnostic method addresses the inefficiencies in the existing methods for testing these viruses.
Cite This Article
APA
Mori A, De Benedictis P, Marciano S, Zecchin B, Zuin A, Zecchin B, Capua I, Cattoli G.
(2008).
Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens.
J Virol Methods, 155(2), 175-181.
https://doi.org/10.1016/j.jviromet.2008.10.009 Publication
Researcher Affiliations
- Istituto Zooprofilattico Sperimentale delle Venezie, Research & Development Department, Viale dell'Universita' 10, 35020 Legnaro, Padova, Italy.
MeSH Terms
- Animals
- DNA Primers
- Erbovirus / genetics
- Erbovirus / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Picornaviridae / genetics
- Picornaviridae / isolation & purification
- Picornaviridae Infections / diagnosis
- Picornaviridae Infections / veterinary
- Picornaviridae Infections / virology
- Polymerase Chain Reaction / methods
- Reproducibility of Results
- Rhinitis / veterinary
- Rhinitis / virology
- Sensitivity and Specificity
- Taq Polymerase
Citations
This article has been cited 3 times.- Woo PC, Lau SK, Choi GK, Huang Y, Wernery R, Joseph S, Wong EY, Elizabeth SK, Patteril NA, Li T, Wernery U, Yuen KY. Equine rhinitis B viruses in horse fecal samples from the Middle East. Virol J 2016 Jun 7;13:94.
- Lu Z, Timoney PJ, White J, Balasuriya UB. Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses. BMC Vet Res 2012 Jul 25;8:120.
- Stasiak K, Dunowska M, Rola J. Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland. Viruses 2024 Jul 26;16(8).
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