Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan.
Abstract: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). Unassigned: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. Unassigned: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 10 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. Unassigned: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.
Copyright: © Sandybayev, et al.
Publication Date: 2023-08-19 PubMed ID: 37766711PubMed Central: PMC10521171DOI: 10.14202/vetworld.2023.1682-1689Google Scholar: Lookup
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Summary
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The research study highlights the development and testing of a new real-time polymerase chain reaction (RT-PCR) assay system used in the detection of Equine Influenza Virus (EIV) subtype H3. This system aims to improve the diagnosis and surveillance of equine influenza, a severe infectious disease in horses, in Kazakhstan.
Research Methodology
- The RT-PCR assay evaluation included five EIV strains as positive controls and ten nasopharyngeal swab samples from horses. They used RNA isolated using GeneJet Viral DNA and RNA Purification Kit.
- The team created primers and probes with the Integrated DNA Technology PrimerQuest Tool for use in the RT-PCR assay.
- The researchers optimized the assay through analysis of the annealing temperature and primer and probes concentrations. They also evaluated its sensitivity and specificity.
- Sequencing was carried out with Thermo Fisher 3130 Genetic Analyzer and the evolutionary history inferred using the Neighbor-Joining method.
Outcome
- The researchers found that the primers and probes they designed, which target the H3 gene, are specific to the EIV.
- The RT-PCR assay could detect as low as 50 femtogram (f) or 3 × 10 copies of genomic RNA, thereby demonstrating high sensitivity.
- No cross-reactions with other respiratory viral and bacterial pathogens were observed, indicating high specificity.
Testing & Validation
- To ensure its effectiveness, ten nasopharyngeal swab samples from farms in North Kazakhstan regions were analyzed.
- The accuracy of the assay was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for Equine Influenza identification.
Conclusion and Impact
- The research showed that the developed RT-PCR assay has high sensitivity and specificity for detecting EIV.
- This easy-to-use, highly sensitive, and specific assay is a reliable tool for diagnosing and surveilling EI.
- Its implementation in veterinary practice will enable faster response to potential EI outbreaks, thus resulting in better health outcomes for horses in Kazakhstan.
Cite This Article
APA
Sandybayev N, Strochkov V, Beloussov V, Orkara S, Kydyrmanov A, Khan Y, Batanova Z, Kassenov M.
(2023).
Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan.
Vet World, 16(8), 1682-1689.
https://doi.org/10.14202/vetworld.2023.1682-1689 Publication
Researcher Affiliations
- Kazakhstan-Japan Innovation Centre, Kazakh National Agrarian Research University, 050010 Almaty, Kazakhstan.
- Kazakhstan-Japan Innovation Centre, Kazakh National Agrarian Research University, 050010 Almaty, Kazakhstan.
- TreeGene Molecular Genetics Laboratory, Almaty 050009, Kazakhstan.
- Kazakhstan-Japan Innovation Centre, Kazakh National Agrarian Research University, 050010 Almaty, Kazakhstan.
- Research and Production Center for Microbiology and Virology, Almaty 050060, Kazakhstan.
- Research and Production Center for Microbiology and Virology, Almaty 050060, Kazakhstan.
- Faculty of Veterinary, Kazakh National Agrarian Research University, Almaty 050010, Kazakhstan.
- Laboratory of Virology, Kazakh Scientific Research Veterinary Institute, Almaty 050016, Kazakhstan.
Conflict of Interest Statement
The authors declare that they have no competing interests.
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