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Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.
Abstract: Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.
Publication Date: 2008-03-06 PubMed ID: 18319426DOI: 10.1177/104063870802000202Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The researchers compared two specific types of genetic detection methods for finding a virus called Equine arteritis virus (EAV) in horse semen and lab-grown cells. They found that one method was more sensitive for detecting the virus in horse semen, but it was less sensitive than the standard cell culture technique. Both methods were highly effective for detecting the virus in lab-grown cells. The researchers suggest confirmation tests are still needed for certain cases, and stress the importance of comparing and validating testing methods.
Comparison of Two RT-PCR Assays
- The researchers compared two 1-tube real-time reverse transcription polymerase chain reaction (RT-PCR) assays, called T1 and T2, for detecting Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF).
- Competing against the traditional method of virus isolation (VI) in cell culture, the assays were tested for their specificity (how well they detect only the intended target), and sensitivity (how well they can detect low amounts of the target).
Sensitivity and Specificity of the Assays
- The T1 was more sensitive than the T2 with 93.4% versus 42.6% sensitivity for detection of EAV RNA in semen.
- However, the T1 assay was less sensitive than the gold standard VI test as prescribed by the World Organization for Animal Health (OIE).
- Both PCR assays showed high sensitivity levels when detecting EAV RNA in TCF, with T1 having a full 100% sensitivity and T2 following close behind with a 95.2% sensitivity.
Implications of the Findings
- The study points out discrepancies in sensitivity between these RT-PCR assays and the VI test, particularly noting that horse semen samples from EAV-seropositive stallions that test negative for EAV by the RT-PCR assays should be further confirmed free of virus by the VI method.
- Similarly, TCF samples that are VI-positive but RT-PCR negative should also be confirmed using a one-way neutralization test or fluorescent antibody test specifically targeting EAV.
- If these further tests confirm the absence of EAV, such samples should then be tested for the presence of other equine viral pathogens to ascertain what other viruses may be present.
- This study highlights the importance of making comparative evaluations and validations of real-time RT-PCR assays before their use in diagnostic settings for the detection and identification of specific infectious agents.
Cite This Article
APA
Lu Z, Branscum AJ, Shuck KM, Zhang J, Dubovi EJ, Timoney PJ, Balasuriya UB.
(2008).
Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.
J Vet Diagn Invest, 20(2), 147-155.
https://doi.org/10.1177/104063870802000202 Publication
Researcher Affiliations
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA.
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Arterivirus Infections / veterinary
- Arterivirus Infections / virology
- Equartevirus / genetics
- Equartevirus / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Male
- RNA, Viral / analysis
- RNA, Viral / chemistry
- RNA, Viral / genetics
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Semen / virology
- Sensitivity and Specificity
Citations
This article has been cited 14 times.- Thieulent CJ, Carossino M, Peak L, Wolfson W, Balasuriya UBR. Development and validation of multiplex one-step qPCR/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and pathogens associated with feline respiratory disease complex. PLoS One 2024;19(3):e0297796.
- Agerholm JS, Klas EM, Damborg P, Borel N, Pedersen HG, Christoffersen M. A Diagnostic Survey of Aborted Equine Fetuses and Stillborn Premature Foals in Denmark. Front Vet Sci 2021;8:740621.
- Okuyama T, Ohtsuka K, Ogura W, Yonetani S, Yamasaki S, Miyagi H, Sekiguchi K, Ohnishi H, Watanabe T. Establishing an internal quality control method for the stable extraction of nucleic acids of severe acute respiratory syndrome coronavirus 2 and RT-PCR-based detection. J Clin Lab Anal 2021 Nov;35(11):e23998.
- Go YY, Kim YS, Cheon S, Nam S, Ku KB, Kim M, Cho NH, Park H, Alison Lee PY, Lin YC, Tsai YL, Thomas Wang HT, Balasuriya UBR. Evaluation and Clinical Validation of Two Field-Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome-Coronavirus. J Mol Diagn 2017 Nov;19(6):817-827.
- Go YY, Rajapakse RPVJ, Kularatne SAM, Lee PA, Ku KB, Nam S, Chou PH, Tsai YL, Liu YL, Chang HG, Wang HT, Balasuriya UBR. A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer. J Clin Microbiol 2016 Jun;54(6):1528-1535.
- Back H, Penell J, Pringle J, Isaksson M, Ronéus N, Treiberg Berndtsson L, Ståhl K. A longitudinal study of poor performance and subclinical respiratory viral activity in Standardbred trotters. Vet Rec Open 2015;2(1):e000107.
- Eschbaumer M, Li WM, Wernike K, Marshall F, Czub M. Probe-free real-time reverse transcription polymerase chain reaction assays for the detection and typing of porcine reproductive and respiratory syndrome virus in Canada. Can J Vet Res 2015 Jul;79(3):170-9.
- Balasuriya UB, Go YY, MacLachlan NJ. Equine arteritis virus. Vet Microbiol 2013 Nov 29;167(1-2):93-122.
- 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.
- Zhang J, Go YY, Huang CM, Meade BJ, Lu Z, Snijder EJ, Timoney PJ, Balasuriya UB. Development and characterization of an infectious cDNA clone of the modified live virus vaccine strain of equine arteritis virus. Clin Vaccine Immunol 2012 Aug;19(8):1312-21.
- Miszczak F, Shuck KM, Lu Z, Go YY, Zhang J, Sells S, Vabret A, Pronost S, Fortier G, Timoney PJ, Balasuriya UB. Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen. J Clin Microbiol 2011 Oct;49(10):3694-6.
- Go YY, Zhang J, Timoney PJ, Cook RF, Horohov DW, Balasuriya UB. Complex interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes. J Virol 2010 May;84(10):4898-911.
- Zhang J, Stein DA, Timoney PJ, Balasuriya UB. Curing of HeLa cells persistently infected with equine arteritis virus by a peptide-conjugated morpholino oligomer. Virus Res 2010 Jun;150(1-2):138-42.
- Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus. J Clin Microbiol 2009 Dec;47(12):3907-13.
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