Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.
Abstract: Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.
Copyright © 2016 Elsevier B.V. All rights reserved.
Publication Date: 2016-12-16 PubMed ID: 27993615DOI: 10.1016/j.jviromet.2016.12.010Google Scholar: Lookup
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Summary
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The research article is about the development and evaluation of a new, cost-effective method to detect equine herpesvirus-1 (EHV-1), a major problem for the US horse industry. The method involves an insulated isothermal PCR (iiPCR) assay, comparable in sensitivity and specificity to previously used real-time PCR assays.
Background of the Study
- The study deals with the challenge of diagnosing Equine Herpesvirus Myeloencephalopathy (EHM), a significant issue for the US equine industry, caused by the EHV-1 virus. This virus is associated with a variety of conditions in horses, including upper respiratory disease, abortion, and chorioretinal lesions.
Goal of the Study
- The research aims to develop an economical and user-friendly insulated isothermal PCR (iiPCR) method for detecting EHV-1. The method targets the Open Reading 30 (ORF30), a region in the virus’s genetic material, to detect both the neuropathogenic and non-neuropathogenic strains of the virus. This detection method was developed for use with the POCKIT™ device, a field-deployable tool for point-of-need detection of EHV-1.
Methodology and Results
- The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction, signifying it could identify the presence of the virus even if few viral particles were present.
- The iiPCR assay was designed and tested to ensure that it did not cross-react with ten non-target equine viral pathogens, proving the assay’s specificity in detecting only EHV-1.
- The performance of the novel EHV-1 iiPCR assay was compared to two existing real-time PCR (qPCR) assays using 104 archived clinical samples, in two independent laboratories.
- Results demonstrated a high level of agreement between the iiPCR and the qPCR methods. Out of 53 qPCR-positive samples, all were also found positive by the iiPCR assay. Similarly, 46 of the 51 samples that tested negative using qPCR were also negative using the iiPCR assay.
- An overall agreement of 95.19% was observed between the two assays, demonstrating the robustness and reliability of the new iiPCR method.
Conclusion
- The research concluded that the new EHV-1 iiPCR assay is robust, providing sensitivity and specificity that are comparable to the existing qPCR assays for the detection of EHV-1 nucleic acid in clinical samples.
Cite This Article
APA
Balasuriya UB, Lee PA, Tsai YL, Tsai CF, Shen YH, Chang HG, Skillman A, Wang HT, Pronost S, Zhang Y.
(2016).
Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.
J Virol Methods, 241, 58-63.
https://doi.org/10.1016/j.jviromet.2016.12.010 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. Electronic address: ubalasuriya@uky.edu.
- GeneReach USA, Lexington, MA, USA. Electronic address: peiyu329@genereachbiotech.com.
- GeneReach USA, Lexington, MA, USA. Electronic address: fiwind.tsai@genereachbiotech.com.
- GeneReach USA, Lexington, MA, USA. Electronic address: marktsai@genereachbiotech.com.
- GeneReach USA, Lexington, MA, USA. Electronic address: yuki7510zz@genereachbiotech.com.
- GeneReach USA, Lexington, MA, USA. Electronic address: gracechang@genereachbiotech.com.
- Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA. Electronic address: ashley.skillman@uky.edu.
- GeneReach USA, Lexington, MA, USA. Electronic address: thomaswang@genereachbiotech.com.
- LABÉO(-)Frank Duncombe, U2RM, EA4655 Normandie Université, UNICAEN, 1 Route de Rosel, 14053 Caen, France. Electronic address: Stephane.PRONOST@laboratoire-labeo.fr.
- Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH, USA. Electronic address: YZhang@agri.ohio.gov.
MeSH Terms
- Animals
- DNA, Viral / genetics
- DNA, Viral / isolation & purification
- Encephalomyelitis / diagnosis
- Encephalomyelitis / veterinary
- Encephalomyelitis / virology
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Open Reading Frames / genetics
- Polymerase Chain Reaction / economics
- Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Temperature
Citations
This article has been cited 8 times.- Nielsen SS, Alvarez J, Bicout DJ, Calistri P, Canali E, Drewe JA, Garin-Bastuji B, Gonzales Rojas JL, Gortázar C, Herskin M, Michel V, Miranda Chueca MÁ, Roberts HC, Padalino B, Pasquali P, Spoolder H, Ståhl K, Calvo AV, Viltrop A, Winckler C, Carvelli A, Paillot R, Broglia A, Kohnle L, Baldinelli F, Van der Stede Y. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Equine Herpesvirus-1. EFSA J 2022 Jan;20(1):e07036.
- Chu H, Liu C, Liu J, Yang J, Li Y, Zhang X. Recent advances and challenges of biosensing in point-of-care molecular diagnosis. Sens Actuators B Chem 2021 Dec 1;348:130708.
- Knox A, Beddoe T. Isothermal Nucleic Acid Amplification Technologies for the Detection of Equine Viral Pathogens. Animals (Basel) 2021 Jul 20;11(7).
- 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.
- Tsai JJ, Liu WL, Lin PC, Huang BY, Tsai CY, Chou PH, Lee FC, Ping CF, Lee PA, Liu LT, Chen CH. An RT-PCR panel for rapid serotyping of dengue virus serotypes 1 to 4 in human serum and mosquito on a field-deployable PCR system. PLoS One 2019;14(3):e0214328.
- 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.
- Petano-Duque JM, Urueña-Martinez E, Cabezas-Callejas LL, Perilla-Amaya J, Rueda-García V, Rondón-Barragán IS, Lopera-Vásquez R. Molecular and Serological Investigation of Equine Herpesvirus Type 1 (EHV-1) and Type 4 (EHV-4) in Horses In Ibagué, Tolima. Vet Med Int 2025;2025:1661949.
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