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Journal of clinical microbiology2009; 47(12); 3907-3913; doi: 10.1128/JCM.00598-09

Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.

Abstract: The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.
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Publication Date: 2009-10-21 PubMed ID: 19846644PubMed Central: PMC2786649DOI: 10.1128/JCM.00598-09Google Scholar: Lookup
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  • Evaluation Study
  • 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.

This research article discusses the development and testing of new detection assays for equine influenza virus (EIV) using TaqMan real-time reverse transcription-PCR (rRT-PCR). The study comes up with eight assays and confirms that they do not mix reactions with other known equine respiratory viruses. For testing, three rRT-PCR assays were selected and used on nasal swabs from horses, showing a higher degree of sensitivity and specificity compared to other testing methods. One assay, in particular, is singled out for its potential to aid surveillance efforts for a specific EIV subtype thought to be extinct or at low levels.

Objectives and Methodology

  • The main aim of this research was to create and test new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays, leveraging minor groove binding probe.
  • These assays were designed for detecting a wide range of Equine Influenza Virus (EIV), comprising both the H3N8 and H7N7 subtypes.
  • In total, eight rRT-PCR assays were created, which targeted the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of both EIV subtypes.

Assay Evaluation

  • None of the assays developed showed a cross-reaction with any other known equine respiratory viruses, proving their specificity for EIV.
  • For testing and evaluation, three rRT-PCR assays, EqFlu NP, M, and HA3, were used on nasal swabs taken from horses either for routine diagnosis or experimental inoculation.
  • These three assays showed greater sensitivity (93%, 89%, and 87% respectively) and specificity than virus isolation through egg inoculation methods.

Comparison and Findings

  • Sensitivities of rRT-PCR assays that targeted NP and M genes on both EIV subtypes were compared with egg inoculation and the Directigen Flu A test. The comparison showed that molecular assays gave the highest sensitivity.
  • The EqFlu HA7 assay that targets the H7 HA gene showed high specificity for the H7N7 subtype of EIV.
  • This particular subtype is believed to be extinct, or perhaps circulating at a very low level. As such, this assay could be instrumental for reliable surveillance of this subtype.

Conclusion

  • The new assays developed in this study offer a reliable, rapid means for EIV diagnosis, as well as identification of EIV subtypes, thus significantly improving detection capabilities.

Cite This Article

APA
Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. (2009). Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus. J Clin Microbiol, 47(12), 3907-3913. https://doi.org/10.1128/JCM.00598-09

Publication

Journal of clinical microbiology
ISSN: 1098-660X
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 47
Issue: 12
Pages: 3907-3913

Researcher Affiliations

Lu, Zhengchun
  • Department of Veterinary Science, University of Kentucky, Maxwell H Gluck Equine Research Center, Lexington, Kentucky 40546, USA.
Chambers, Thomas M
    Boliar, Saikat
      Branscum, Adam J
        Sturgill, Tracy L
          Timoney, Peter J
            Reedy, Stephanie E
              Tudor, Lynn R
                Dubovi, Edward J
                  Vickers, Mary Lynne
                    Sells, Stephen
                      Balasuriya, Udeni B R

                        MeSH Terms

                        • Animals
                        • Chick Embryo
                        • Hemagglutinin Glycoproteins, Influenza Virus / genetics
                        • Horse Diseases / diagnosis
                        • Horse Diseases / virology
                        • Horses
                        • Influenza A Virus, H3N8 Subtype / classification
                        • Influenza A Virus, H3N8 Subtype / genetics
                        • Influenza A Virus, H3N8 Subtype / isolation & purification
                        • Influenza A Virus, H7N7 Subtype / classification
                        • Influenza A Virus, H7N7 Subtype / genetics
                        • Influenza A Virus, H7N7 Subtype / isolation & purification
                        • Nucleoproteins / genetics
                        • Orthomyxoviridae Infections / veterinary
                        • Orthomyxoviridae Infections / virology
                        • Reverse Transcriptase Polymerase Chain Reaction / methods
                        • Sensitivity and Specificity
                        • Taq Polymerase
                        • Viral Matrix Proteins / genetics

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                        This article has been cited 16 times.
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