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Viruses2023; 15(8); 1626; doi: 10.3390/v15081626

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.

Abstract: Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission.
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Publication Date: 2023-07-26 PubMed ID: 37631969PubMed Central: PMC10459720DOI: 10.3390/v15081626Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.
  • Research Support
  • Non-U.S. Gov't

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.

The research focuses on refining diagnostic tools for differentiating between equine rotavirus A and B and identifying prevalent genotypes for better infectious disease control in horses and potentially, humans. The study presents a developed quadruplex TaqMan RT-qPCR assay and evaluates its effectiveness compared to previous methods.

About the Research

  • The research primarily targets equine rotavirus A (ERVA) and equine rotavirus B (ERVB), two agents causing significant diarrheal disease in foals. Recently, equine G3-like RVA, a strain of ERVA, was identified as an emerging infection in children.
  • As ERVB has also started to emerge as a cause of foal diarrhea in the US, it was essential for the research to adapt diagnostic methods to improve detection and surveillance of these emerging strains. The data gathered from improved surveillance methods can also inform future vaccine development.
  • The researchers developed a test – the quadruplex TaqMan RT-qPCR assay. This test is specifically designed to differentiate between ERVA and ERVB and identify the types of ERVA strains.

Methods and Findings

  • The research evaluated the analytical and clinical performance of the new quadruplex TaqMan RT-qPCR assay and compared it to two other tests: a previously established ERVA triplex RT-qPCR assay and a standard RT-PCR assay (combined with Sanger sequencing of PCR products).
  • This quadruplex RT-qPCR assay demonstrated high sensitivity (over 90%) and specificity (100%) for every target, and overall agreement above 96%. This indicates that the test reliably detected and differentiated between ERVA and ERVB, performing better than or on par with previous methods.
  • A comparison between the triplex and quadruplex assays revealed a slightly higher sensitivity for the ERVA NSP3 target using the triplex format. No significant differences were detected for other targets, proving the effectiveness of the quadruplex RT-qPCR assay.

Significance and Future Implications

  • This developed assay will significantly enhance the rapid surveillance of circulating and emerging strains of both ERVA and ERVB. This is crucial for their timely detection and corresponding disease control efforts.
  • The research outcome could potentially impact both veterinary and human medicine, given the detected interspecies transmission of certain strains.
  • Promising advancements such as these in molecular diagnostic tools contribute to effective disease management in horses, and potentially humans, improving health and wellbeing.

Cite This Article

APA
Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V. (2023). 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, 15(8), 1626. https://doi.org/10.3390/v15081626

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 15
Issue: 8
PII: 1626

Researcher Affiliations

Carossino, Mariano
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Balasuriya, Udeni B R
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Thieulent, Côme J
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Barrandeguy, Maria E
  • Escuela de Veterinaria, Universidad del Salvador, Buenos Aires B1630, Argentina.
  • Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires B1686, Argentina.
Vissani, Maria Aldana
  • Escuela de Veterinaria, Universidad del Salvador, Buenos Aires B1630, Argentina.
  • Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires B1686, Argentina.
  • Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1425, Argentina.
Parreño, Viviana
  • Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires B1686, Argentina.
  • Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1425, Argentina.

MeSH Terms

  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / veterinary
  • Rotavirus / isolation & purification
  • Animals
  • Horses
  • Horse Diseases / virology
  • Rotavirus Infections / veterinary
  • Feces / virology
  • Sensitivity and Specificity

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

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Citations

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