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Home / Equine Research Bank / Microorganisms / Designing a Multiplex PCR-xMAP Assay for the Detection and D...
Microorganisms2024; 12(5); 932; doi: 10.3390/microorganisms12050932

Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9.

Abstract: African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4-6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide.
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Publication Date: 2024-05-03 PubMed ID: 38792762PubMed Central: PMC11124020DOI: 10.3390/microorganisms12050932Google Scholar: Lookup
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  • Journal Article

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.

Overview

  • This study developed a new molecular assay combining multiplex PCR and xMAP microsphere technology for the rapid detection and differentiation of all nine African horse sickness virus (AHSV) serotypes in a single test.
  • The assay provides quick, sensitive, and specific identification, supporting better disease management, vaccine selection, and epidemiological tracking of African horse sickness outbreaks.

Background

  • African horse sickness (AHS) is a deadly viral disease affecting horses and other equids globally, causing severe illness and often death.
  • The disease is caused by African horse sickness virus (AHSV), which has nine distinct serotypes (variants).
  • Identifying the specific serotype involved in an outbreak is crucial because it influences treatment strategies, vaccine choice, and helps trace the source and spread of the disease.
  • Current diagnostic methods may be time-consuming or limited in simultaneously detecting multiple serotypes.

Objective

  • The research aimed to develop and optimize a rapid, multiplex molecular assay that could detect and differentiate all nine AHSV serotypes in a single test run.
  • Key goals included improving diagnostic speed, sensitivity, specificity, and throughput, making the assay practical for large-scale epidemiological use.

Methodology

  • Multiplex PCR: A technique that amplifies multiple DNA targets simultaneously by using several primer sets in one reaction, enabling detection of multiple serotypes in one assay.
  • xMAP (microsphere-based technology): Uses microspheres (tiny beads) tagged with specific probes to hybridize with PCR products, allowing highly specific identification of each serotype based on bead fluorescence.
  • The assay was designed to target unique genetic regions specific to each AHSV serotype to ensure accurate differentiation.
  • Optimization involved adjusting primer concentrations, reaction conditions, and hybridization parameters to maximize detection efficiency.

Results

  • The assay successfully identified all nine AHSV serotypes with varied detection limits, ranging from as low as 1 genome copy per microliter to 277 copies per microliter depending on the serotype.
  • Diagnostic performance evaluation showed:
    • Sensitivity: 88% – ability of the test to correctly identify positive samples.
    • Specificity: 100% – ability of the test to correctly identify negative samples.
  • The assay can process up to 42 samples simultaneously.
  • The total time from sample to result was approximately 4 to 6 hours, allowing for rapid diagnostic turnaround.

Significance and Applications

  • The development of this assay provides an advanced diagnostic tool that enhances the rapid detection and serotyping of AHSV.
  • Rapid serotype identification helps veterinarians and researchers make informed decisions about vaccination strategies and disease control.
  • High-throughput capability enables large-scale screening during outbreaks, facilitating better epidemiological data collection.
  • This assay can aid in monitoring disease incidence, tracing infection routes, and supporting global efforts to control and manage African horse sickness effectively.

Conclusion

  • This novel multiplex PCR-xMAP assay represents a significant improvement in the molecular diagnosis of African horse sickness virus.
  • It combines sensitivity, specificity, speed, and multiplexing capacity to offer a practical tool for veterinary diagnostic labs and disease surveillance programs worldwide.

Cite This Article

APA
Ashby M, Moore R, King S, Newbrook K, Flannery J, Batten C. (2024). Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9. Microorganisms, 12(5), 932. https://doi.org/10.3390/microorganisms12050932

Publication

Microorganisms
ISSN: 2076-2607
NlmUniqueID: 101625893
Country: Switzerland
Language: English
Volume: 12
Issue: 5
PII: 932

Researcher Affiliations

Ashby, Martin
  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
Moore, Rebecca
  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
King, Simon
  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
Newbrook, Kerry
  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
Flannery, John
  • Department of Pharmaceutical Sciences and Biotechnology, Technological University of the Shannon, Athlone Campus, N37HD68 Athlone, Ireland.
Batten, Carrie
  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.

Conflict of Interest Statement

The authors declare no conflicts of interest.

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