Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.
Abstract: A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time TaqMan PCR assay detected EAV RNA in all samples that were confirmed to contain infectious EAV by virus isolation. The assay had an analytical sensitivity of 10 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per ml. Thus, the one-tube real-time TaqMan RT-PCR assay provides a rapid, accurate, quantitative, convenient and high sample throughput system for diagnosis of EAV infection, in a closed-tube format that minimizes the risk of cross-contamination.
Publication Date: 2002-02-19 PubMed ID: 11849680DOI: 10.1016/s0166-0934(01)00416-5Google Scholar: Lookup
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- Comparative Study
- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article discusses the development and validation of a rapid, accurate, and highly sensitive diagnostic test for detecting Equine Arteritis Virus (EAV) using a one-tube real-time TaqMan Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay.
Development of the RT-PCR Assay
- The one-tube real-time TaqMan RT-PCR assay was designed specifically for the detection of EAV.
- The test used primers and a fluorogenic TaqMan probe to amplify and identify a highly conserved region of the open reading frame 7 (ORF7) of EAV. This area of the viral genome was targeted due to its consistent presence across diverse strains of the virus.
Validation of the Assay
- The validation process involved testing the assay on seminal plasma and nasal secretions of horses that were known to be infected with EAV. These samples contained active, infectious versions of the virus, as confirmed by traditional virus isolation methods in rabbit kidney thirteen (RK-13) cells.
- The test was also applied on a range of cell culture-propagated strains of EAV from Europe and North America to ensure its efficacy across diverse EAV strains.
- The RT-PCR assay was able to identify EAV RNA in all samples that were confirmed to contain the virus through traditional isolation methods, effectively demonstrating the assay’s high degree of accuracy.
Characteristics and Advantages of the Assay
- The RT-PCR assay was found to possess high analytical sensitivity, with a capacity to detect as few as 10 molecules of EAV RNA. This allows the diagnosis of EAV infection in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per milliliter.
- The assay offers a quick, precise, and quantifiable system for diagnosing EAV infection. This improves upon current systems by offering a high sample throughput, meaning it can process numerous samples efficiently.
- Furthermore, the one-tube, closed-tube format of the assay was designed to minimize the risk of cross-contamination, making it a more secure and reliable method for virus detection.
Cite This Article
APA
Balasuriya UB, Leutenegger CM, Topol JB, McCollum WH, Timoney PJ, MacLachlan NJ.
(2002).
Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.
J Virol Methods, 101(1-2), 21-28.
https://doi.org/10.1016/s0166-0934(01)00416-5 Publication
Researcher Affiliations
- Bernard and Gloria Salick Equine Viral Disease Laboratory, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, One Shields Avenue, University of California, Davis, CA 95616, USA. ubbalasuriya@ucdavis.edu
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Arterivirus Infections / veterinary
- Cell Line
- Conserved Sequence
- Equartevirus / genetics
- Equartevirus / isolation & purification
- Europe
- Horse Diseases / virology
- Horses
- Male
- Nasal Mucosa / metabolism
- Nasal Mucosa / virology
- North America
- Open Reading Frames
- RNA, Viral / analysis
- Rabbits
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Semen / virology
- Sensitivity and Specificity
- Taq Polymerase / chemistry
- Time Factors
Citations
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