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BMC veterinary research2012; 8; 120; doi: 10.1186/1746-6148-8-120

Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses.

Abstract: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. Results: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). Conclusions: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.
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Publication Date: 2012-07-25 PubMed ID: 22830930PubMed Central: PMC3542198DOI: 10.1186/1746-6148-8-120Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

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This research paper discusses the development of molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) aimed at detecting and distinguishing between equine rhinitis viruses A and B (ERAV and ERBV), overcoming the challenges of traditional laboratory diagnosis methods for these infections.

Background and Aim

  • The research sets out to enhance the diagnosis of ERAV and ERBV infections in horses, which are prevalent in many countries and currently diagnosed chiefly via virus isolation (VI). This method experiences varying success due to differences in lab practice, incomplete viral growth in cell cultures, and lack of cytopathic effect.
  • The intent of the study was to create molecular diagnostic assays for detecting and identifying ERAV from ERBV, thereby resolving the issues traditionally associated with these infections’ laboratory diagnosis.

Methods and Experimental Design

  • Three rRT-PCR assays aimed at the 5′-UTR of ERAV and ERBV were developed. One was specific for ERAV, and the remaining two were ERBV-specific. Similarly, six cRT-PCR assays were designed targeting the 5′-UTR and 3D polymerase areas of both ERAV and ERBV.
  • These rRT-PCR and cRT-PCR assays were evaluated using RNA derived from 21 archived tissue culture fluid (TCF) samples, previously confirmed as positive for ERAV or ERBV with mono-specific rabbit antisera.

Results

  • The results revealed that the ERAV-specific rRT-PCR and cRT-PCR assays could only detect ERAV isolates, not ERBV. Similarly, ERBV-specific rRT-PCR and cRT-PCR assays could exclusively detect ERBV isolates and not ERAV.
  • Crucially, none of these assays cross-reacted with other prevalent equine respiratory viruses, assuring their specificity.
  • The detection limit for almost all these assays was established at 1 plaque forming unit per milliliter (pfu/ml).

Conclusions

  • The new rRT-PCR and cRT-PCR assays offer an improved diagnostic capability for the detection and differentiation of ERAV and ERBV. They promise to provide a more reliable alternative to the traditional virus isolation method, ensuring dependable detection of ERAV and ERBV in horses.’s respiratory infections.
  • However, a larger number of clinical specimens must be tested before these assays can be fully validated for identifying ERAV and/or ERBV in suspected cases of either viral infection.

Cite This Article

APA
Lu Z, Timoney PJ, White J, Balasuriya UB. (2012). Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses. BMC Vet Res, 8, 120. https://doi.org/10.1186/1746-6148-8-120

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 8
Pages: 120

Researcher Affiliations

Lu, Zhengchun
  • Maxwell H, Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, 108 Maxwell H, Lexington, KY 40546, USA.
Timoney, Peter J
    White, Jena
      Balasuriya, Udeni Br

        MeSH Terms

        • Animals
        • Aphthovirus / genetics
        • Aphthovirus / isolation & purification
        • Cell Line
        • Erbovirus / genetics
        • Erbovirus / isolation & purification
        • Rabbits
        • Real-Time Polymerase Chain Reaction / methods
        • Real-Time Polymerase Chain Reaction / veterinary
        • Reverse Transcriptase Polymerase Chain Reaction / methods
        • Reverse Transcriptase Polymerase Chain Reaction / veterinary
        • Sensitivity and Specificity
        • Serotyping

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        Citations

        This article has been cited 3 times.
        1. Uchida-Fujii E, Kinoshita Y, Niwa H, Maeda T, Nukada T, Ueno T. High prevalence of Mycoplasma equirhinis in Thoroughbred horses with respiratory symptoms in autumn 2018. J Vet Med Sci 2021 Dec 9;83(12):1907-1912.
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        2. Ambagala A, Fisher M, Goolia M, Nfon C, Furukawa-Stoffer T, Ortega Polo R, Lung O. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus. Transbound Emerg Dis 2017 Oct;64(5):1610-1623.
          doi: 10.1111/tbed.12554pubmed: 27589902google scholar: lookup
        3. Stasiak K, Dunowska M, Rola J. Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland. Viruses 2024 Jul 26;16(8).
          doi: 10.3390/v16081204pubmed: 39205178google scholar: lookup
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