Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.
Abstract: Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.
Copyright © 2016 Elsevier B.V. All rights reserved.
Publication Date: 2016-03-29 PubMed ID: 27036504DOI: 10.1016/j.jviromet.2016.02.015Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Diagnosis
- Diagnostic Technique
- Disease control
- Disease Diagnosis
- Disease Management
- Disease Outbreaks
- Disease Prevention
- Disease Surveillance
- Disease Transmission
- Disease Treatment
- Equine Diseases
- Equine Health
- Equine Research
- Equine Science
- Equine Viral Arteritis
- Infection
- Polymerase Chain Reaction
- Real-Time PCR
- Veterinary Medicine
- Veterinary Research
- Virus
Summary
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The research article discusses the development and effectiveness of a method, known as RT-iiPCR, for detecting equine arteritis virus (EAV), a respiratory and reproductive disease in horses that can lead to abortions in pregnant mares, in equine semen and tissue samples. This newly developed method was found to be very sensitive and accurate in identifying EAV, outperforming the previously used real-time RT-PCR technique, especially when used on tissue samples.
The Study
- The researchers aimed to develop and assess a method called reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the identification of Equine arteritis virus (EAV) in equine semen and tissue samples.
- EAV causes equine viral arteritis (EVA), a respiratory and reproductive disease affecting horses.
- Key concern with EAV is that it triggers abortions in pregnant mares and can establish a long-lasting infection in 10-70% of infected stallions, who continue to excrete the virus in their semen.
- The objective was to deliver a test that was more precise in detecting EAV to aid in controlling and preventing the disease.
Findings
- The new RT-iiPCR test showed a 10-fold higher sensitivity than the previously described real-time RT-qPCR.
- The limit of detection was as low as 10 RNA copies.
- When evaluated on 125 semen samples, the sensitivity and specificity of the new RT-iiPCR test were measured as 100.00% and 98.33%, respectively.
- For the old RT-qPCR technique, these values were 98.46% and 100.00% respectively.
- Both assays demonstrated equivalent accuracy in comparison to virus isolation.
- The use of RT-iiPCR on various tissue samples demonstrated a relative sensitivity, specificity, and accuracy of 98.31%, 92.06%, and 95.08%, respectively, outperforming the RT-qPCR technique.
Conclusions
- RT-iiPCR emerges as a highly sensitive, specific, and robust test for the detection of EAV in semen and tissue samples.
- While the older RT-qPCR technique had similar sensitivity and specificity to virus isolation for semen samples, its diagnostic performance proved more limited with tissue samples.
- The introduced RT-iiPCR could be considered as an improved, alternative tool in implementing EAV control and prevention strategies.
Cite This Article
APA
Carossino M, Lee PY, Nam B, Skillman A, Shuck KM, Timoney PJ, Tsai YL, Ma LJ, Chang HF, Wang HT, Balasuriya UB.
(2016).
Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system.
J Virol Methods, 234, 7-15.
https://doi.org/10.1016/j.jviromet.2016.02.015 Publication
Researcher Affiliations
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
- GeneReach USA, Lexington, MA, USA.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.
- GeneReach USA, Lexington, MA, USA.
- GeneReach USA, Lexington, MA, USA.
- GeneReach USA, Lexington, MA, USA.
- GeneReach USA, Lexington, MA, USA.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA. Electronic address: ubalasuriya@uky.edu.
MeSH Terms
- Animals
- Arterivirus Infections / diagnosis
- Arterivirus Infections / prevention & control
- Arterivirus Infections / veterinary
- Arterivirus Infections / virology
- Equartevirus / isolation & purification
- Female
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Male
- Open Reading Frames
- Pregnancy
- RNA, Viral / genetics
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Semen / virology
- Sensitivity and Specificity
- Temperature
Citations
This article has been cited 15 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.
- Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V. Quadruplex Real-Time TaqMan(®) RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes. Viruses 2023 Jul 26;15(8).
- Thieulent CJ, Dittmar W, Balasuriya UBR, Crossland NA, Wen X, Richt JA, Carossino M. Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice. mSphere 2023 Feb 21;8(1):e0055822.
- Chang TD, Huang LN, Lin YJ, Wu ZB, Tsai SH, Lin YH. Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid, Simple DNA Preparation Protocol. Int J Mol Sci 2022 Oct 31;23(21).
- Knox A, Beddoe T. Isothermal Nucleic Acid Amplification Technologies for the Detection of Equine Viral Pathogens. Animals (Basel) 2021 Jul 20;11(7).
- Hemida MG, Waheed M, Ali AM, Alnaeem A. Detection of the Middle East respiratory syndrome coronavirus in dromedary camel's seminal plasma in Saudi Arabia 2015-2017. Transbound Emerg Dis 2020 Nov;67(6):2609-2614.
- Ren Y, Yue H, Zhu L, Tang C, Zhang B. Development and evaluation of reverse transcription-insulated isothermal PCR assay to detect duck hepatitis A virus type A in liver samples using the POCKIT(TM) system. J Vet Med Sci 2019 Oct 24;81(10):1533-1539.
- Zhang J, Nfon C, Tsai CF, Lee CH, Fredericks L, Chen Q, Sinha A, Bade S, Harmon K, Piñeyro P, Gauger P, Tsai YL, Wang HT, Lee PA. Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus. BMC Vet Res 2019 May 24;15(1):168.
- Carossino M, Barrandeguy ME, Erol E, Li Y, Balasuriya UBR. Development and evaluation of a one-step multiplex real-time TaqMan(®) RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples. Virol J 2019 Apr 25;16(1):49.
- Cooke KL, Frenzer P, Tucker SJ, Crawford PC, Kirk SK, Levy JK. Rapid Diagnosis of Babesia gibsoni by Point-of-Need Testing by Insulated Isothermal PCR in Dogs at High Risk of Infection. J Vet Intern Med 2018 Jan;32(1):232-235.
- Carossino M, Li Y, Lee PA, Tsai CF, Chou PH, Williams D, Skillman A, Frank Cook R, Brown G, Chang HG, Wang HT, Balasuriya UBR. Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus. BMC Infect Dis 2017 Dec 19;17(1):778.
- 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.
- Carossino M, Loynachan AT, Canisso IF, Cook RF, Campos JR, Nam B, Go YY, Squires EL, Troedsson MHT, Swerczek T, Del Piero F, Bailey E, Timoney PJ, Balasuriya UBR. Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8(+) T and CD21(+) B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract. J Virol 2017 Jul 1;91(13).
- Lin YH, Lin YJ, Chang TD, Hong LL, Chen TY, Chang PF. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4. PLoS One 2016;11(7):e0159681.
- 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.
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