HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA.
Abstract: Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbes chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x10(5) copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary
Publication Date: 2012-12-12 PubMed ID: 23214358DOI: 10.2478/v10181-012-0064-9Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article explores a new method for detecting equine herpesvirus type 2 (EHV-2) in horses using a HybProbes-based real-time PCR assay. This system shows promise for being a reliable, highly sensitive, and rapid method of virus identification in a veterinary setting.
Understanding Equid Herpesvirus Type 2 (EHV-2)
- The research focuses on EHV-2, a common virus within the Gammaherpesvirinae subfamily affecting horses worldwide.
- EHV-2 has been associated with triggering a latent equid herpesvirus type-1, as incidences of reactivation of EHV-1 have consistently been accompanied by EHV-2 replication.
- The detection and diagnosis of EHV-2 infections have traditionally relied on several techniques ranging from cell culture to PCR methods and modifications of these tools.
Development and Optimization of the Real-Time PCR (qPCR) Assay
- The study’s goal was to create, fine-tune, and validate the specificity of a real-time PCR (qPCR) tool specifically designed to detect EHV-2 DNA through the use of HybProbes chemistry.
- The authors tested the analytic sensitivity of their assay with a variety of viral DNA concentrations (between 70 and 7×10^5 copies/ml).
- Using probit analysis, they determined the limit of detection (LOD) — the lowest quantity of DNA that could still be reliably detected — to be 56 copies/ml.
Evaluation of Clinical Samples
- In their experiment, the researchers tested 20 different clinical samples for the presence of EHV-2, successfully detecting the viral DNA in 25% (5 of 20) of the samples using their custom qPCR method.
- This demonstrated that their HybProbes-based real-time PCR assay is indeed a valuable, reliable tool for the detection and quantification of equid herpesvirus type 2 DNA within diverse clinical samples.
Conclusion
- The speed, accuracy, and high sensitivity of the LightCycler 2.0 instrument, used in this real-time PCR assay, make it an excellent choice for EHV-2 DNA detection in a veterinary setting.
- This study paves the way for faster, more efficient detection and diagnosis of EHV-2, which in turn could lead to more effective treatment strategies for infected horses.
Cite This Article
APA
Osińska E, Golke A, Słońska A, Cymerys J, Bańbura MW, Dzieciatkowski T.
(2012).
HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA.
Pol J Vet Sci, 15(3), 411-416.
https://doi.org/10.2478/v10181-012-0064-9 Publication
Researcher Affiliations
- Interfaculty Department of Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-786 Warsaw, Poland.
MeSH Terms
- Animals
- DNA, Viral / genetics
- DNA, Viral / isolation & purification
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Rhadinovirus / genetics
- Rhadinovirus / isolation & purification
- Sensitivity and Specificity
- Tumor Virus Infections / diagnosis
- Tumor Virus Infections / veterinary
- Tumor Virus Infections / virology
Citations
This article has been cited 2 times.- Cymerys J, Słońska A, Tucholska A, Golke A, Chmielewska A, Bańbura MW. Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons-the possible effects of the multiple passages of EHV-1 on its neurovirulence. Folia Microbiol (Praha) 2018 Jan;63(1):1-11.
- Kim JY, Kang SI, Lee JJ, Lee K, Sung SR, Erdenebaataar J, Vanaabaatar B, Jung SC, Park YH, Yoo HS, Her M. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms. J Vet Med Sci 2016 May 3;78(4):557-62.
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