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Virology journal2019; 16(1); 49; doi: 10.1186/s12985-019-1149-1

Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples.

Abstract: Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. A one-step multiplex TaqMan RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, > 90% for G3 VP7 and > 99% for G14 VP7, respectively) and high overall agreement (> 98%) compared to conventional RT-PCR and sequencing. This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field.
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Publication Date: 2019-04-25 PubMed ID: 31023319PubMed Central: PMC6482509DOI: 10.1186/s12985-019-1149-1Google Scholar: Lookup
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  • Journal Article
  • 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 article presents the development of a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) assay system for rapid detection and genotyping of two prevalent strains of Equine Rotavirus A (ERVA) – G3 and G14 from fecal samples. This new test method appears to be a significant improvement over current genotyping methods, offering high efficiency as well as specificity.

Research Context and Objectives

  • The researchers are addressing the lack of an efficient diagnostic method for ERVA, a common cause of diarrhea in young foals. Given its consequences on equine breeding businesses globally, prompt and accurate detection and characterization of the virus are crucial for control and prevention efforts.
  • The specific aim of this study was to develop a one-step multiplex TaqMan RT-qPCR assay for swift detection and genotyping of ERVA strains G3 and G14, both of which are commonly found in infected foals.

Assay Development and Evaluation

  • The researchers used in vitro transcriptions of RNA containing target sequences to evaluate the sensitivity of the newly developed assay. They used a panel of various group A rotaviruses, as well as other equine viruses and bacteria, to assess its specificity.
  • The researchers compared this new multiplex assay with a standard VP7-specific RT-PCR assay and Sanger sequencing using a panel of 177 fecal samples. They determined its limits of detection, sensitivity, specificity, and agreement.

Results

  • The researchers found the newly developed G3 and G14 VP7 assays to be highly specific and efficient, showing perfect linearity.
  • Although the multiplex assays were 100 times less sensitive than the singleplex assays, this did not impact their clinical performance.
  • The clinical performance of the multiplex RT-qPCR assay revealed its high sensitivity and specificity—100% for NSP3, over 90% for G3 VP7, and over 99% for G14 VP7. It also showed high overall agreement (over 98%) with the standard RT-PCR and sequencing methods.

Conclusions

  • Based on the results, the researchers concluded that this newly developed multiplex RT-qPCR assay could serve as a reliable tool for the rapid detection and genotyping of ERVA strains.
  • The tool aims to aid in controlling and preventing ERVA infections in the field by allowing early detection and accurate characterization of the virus.

Cite This Article

APA
Carossino M, Barrandeguy ME, Erol E, Li Y, Balasuriya UBR. (2019). Development and evaluation of a one-step multiplex real-time TaqMan® RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples. Virol J, 16(1), 49. https://doi.org/10.1186/s12985-019-1149-1

Publication

Virology journal
ISSN: 1743-422X
NlmUniqueID: 101231645
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 49
PII: 49

Researcher Affiliations

Carossino, Mariano
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
  • Escuela de Veterinaria, Universidad del Salvador, Champagnat 1599, Ruta Panamericana km54.5 (B1630AHU), Pilar, Buenos Aires, Argentina.
Barrandeguy, Maria E
  • Escuela de Veterinaria, Universidad del Salvador, Champagnat 1599, Ruta Panamericana km54.5 (B1630AHU), Pilar, Buenos Aires, Argentina.
  • Instituto de Virología, CICVyA, INTA. Las Cabañas y Los Reseros s/n, (1712) Castelar, Buenos Aires, Argentina.
Erol, Erdal
  • Department of Veterinary Science, University of Kentucky Veterinary Diagnostic Laboratory, University of Kentucky, Lexington, KY, USA.
Li, Yanqiu
  • Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Balasuriya, Udeni B R
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA. balasuriya1@lsu.edu.

MeSH Terms

  • Animals
  • Antigens, Viral / genetics
  • Capsid Proteins / genetics
  • Diarrhea / virology
  • Feces / virology
  • Genome, Viral
  • Genotype
  • Genotyping Techniques
  • Horse Diseases / diagnosis
  • Horse Diseases / virology
  • Horses
  • Multiplex Polymerase Chain Reaction / methods
  • Multiplex Polymerase Chain Reaction / standards
  • Phylogeny
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction
  • Rotavirus / genetics
  • Rotavirus / isolation & purification
  • Rotavirus Infections / diagnosis
  • Rotavirus Infections / veterinary
  • Sensitivity and Specificity
  • Viral Nonstructural Proteins / genetics

Grant Funding

  • 1215351520 / Gluck Equine Research Foundation (GERF) competitive grant
  • CVT 123 / INTA-HARAS Agreement

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: Not applicable. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Citations

This article has been cited 11 times.
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