Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.
Abstract: Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses.
Copyright © 2014 Elsevier B.V. All rights reserved.
Publication Date: 2014-06-30 PubMed ID: 24992669DOI: 10.1016/j.jviromet.2014.06.016Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
Summary
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The study presents a new an inexpensive, rapid, and user-friendly technique for the detection of equine influenza virus subtype H3N8 using a method called insulated isothermal RT-PCR (iiRT-PCR) on a portable device called POCKIT™.
About Equine Influenza Virus and its Detection
- Equine influenza is a highly infectious respiratory disease that affects horses. This disease is caused by the Equine Influenza Virus (EIV), and H3N8 is its most significant subtype.
- The rapid diagnosis of this illness is crucial to effectively implement quarantine measures and to control the virus’s spread.
- The study aimed to develop a new technique suitable for point-of-need detection in the field using the insulated isothermal RT-PCR method.
About the Insulated Isothermal RT-PCR Method
- The insulated isothermal RT-PCR (iiRT-PCR) method is a type of polymerase chain reaction that happens at a constant temperature (isothermal).
- The technique was evaluated using in vitro transcribed (IVT) RNA and ten-fold serial dilutions of RNA extracted from the H3N8 strain.
- The iiRT-PCR assay was developed to target the EIV HA3 gene and its sensitivity was analyzed. It was also tested for specificity using inclusivity and exclusivity panels.
Results and Conclusion
- The iiRT-PCR assay was around 100 times more sensitive than reference standard assays in detecting the H3N8 subtype of EIV.
- The assay accurately identified fifteen different strains of EIV H3N8, two strains of Canine Influenza Virus H3N8, and did not cross-react with other subtypes or any other equine respiratory viral pathogens.
- The method showed a 100% agreement in detecting the EIV A/equine/Kentucky/7/07 strain, with 56 nasal swab samples collected from experimentally inoculated horses.
- In conclusion, this new iiRT-PCR assay can provide a reliable, highly sensitive, and specific on-site detection system for Equine and Canine Influenza Viruses when used with a portable POCKIT™ Nucleic Acid Analyzer.
Cite This Article
APA
Balasuriya UB, Lee PY, Tiwari A, Skillman A, Nam B, Chambers TM, Tsai YL, Ma LJ, Yang PC, Chang HF, Wang HT.
(2014).
Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.
J Virol Methods, 207, 66-72.
https://doi.org/10.1016/j.jviromet.2014.06.016 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.
- Department of Research and Development, 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.
- Department of Research and Development, GeneReach USA, Lexington, MA, USA.
- Department of Research and Development, GeneReach USA, Lexington, MA, USA.
- Department of Research and Development, GeneReach USA, Lexington, MA, USA.
- Department of Research and Development, GeneReach USA, Lexington, MA, USA.
- Department of Research and Development, GeneReach USA, Lexington, MA, USA.
MeSH Terms
- Animals
- Horses
- Influenza A Virus, H3N8 Subtype / genetics
- Influenza A Virus, H3N8 Subtype / isolation & purification
- Molecular Diagnostic Techniques / instrumentation
- Molecular Diagnostic Techniques / methods
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / veterinary
- Orthomyxoviridae Infections / virology
- Point-of-Care Systems
- Reverse Transcriptase Polymerase Chain Reaction / instrumentation
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Sensitivity and Specificity
- Veterinary Medicine / instrumentation
- Veterinary Medicine / methods
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