Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses.
Abstract: We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.
Publication Date: 2018-09-21 PubMed ID: 30239276PubMed Central: PMC6505849DOI: 10.1177/1040638718799388Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Treatment
- Equine Diseases
- Equine Health
- Equine Herpesvirus
- Horses
- Infection
- Infectious Disease
- Influenza
- Laboratory Methods
- Polymerase Chain Reaction
- Real-Time PCR
- Respiratory Disease
- Respiratory Health
- Veterinary Medicine
- Veterinary Research
- Virology
- Virus
Summary
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The research article is about the development of a cost-effective and accurate PCR test for the simultaneous detection of major equine respiratory viruses.
Overview of the Research
- The study targeted the development of a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay. This assay was designed for the simultaneous detection of the primary equine respiratory viruses, which include the equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus).
- The assay was specifically made such that the primers and probes would only amplify the targeted viruses. The study confirmed that there were no inter-assay cross-amplifications or nonspecific interactions in this process. This ensures that the test is specific to the targeted viruses alone.
Efficiency of the Assay
- The efficiency of the multiplex assay in detecting and amplifying the viruses was high. It was found to be 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV respectively.
- Moreover, the R values of the monoplex and multiplex assays were equal or greater than 0.990, and the slopes were between -3.37 to -3.59. These statistics confirm the high performance of the assay.
Testing and Validation
- The researchers tested the performance of the assay by analyzing 152 samples collected from clinically infected horses. The EHV-1 DNA was detected in 12 samples, the EHV-4 DNA was detected in 9 samples, and both EHV-1 and EHV-4 were detected in 4 samples.
- The accuracy of the multiplex RT-rtPCR assay was validated by comparing these results with those obtained using commercial real-time PCR and RT-rtPCR kits. This comparison confirmed the accuracy of the developed assay in detecting the targeted viruses.
Conclusion
- The researchers concluded that the developed multiplex RT-rtPCR is a cost-effective, specific, and accurate method for the simultaneous detection of the major equine respiratory viruses, whether they occur alone or as part of coinfections.
Cite This Article
APA
Ghoniem SM, El Deeb AH, Aggour MG, Hussein HA.
(2018).
Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses.
J Vet Diagn Invest, 30(6), 924-928.
https://doi.org/10.1177/1040638718799388 Publication
Researcher Affiliations
- Departments of Virology (Ghoniem), Animal Health Research Institute, Dokki, Egypt.
- Biotechnology (Aggour), Animal Health Research Institute, Dokki, Egypt.
- Department of Virology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt (El Deeb, Hussein).
- Departments of Virology (Ghoniem), Animal Health Research Institute, Dokki, Egypt.
- Biotechnology (Aggour), Animal Health Research Institute, Dokki, Egypt.
- Department of Virology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt (El Deeb, Hussein).
- Departments of Virology (Ghoniem), Animal Health Research Institute, Dokki, Egypt.
- Biotechnology (Aggour), Animal Health Research Institute, Dokki, Egypt.
- Department of Virology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt (El Deeb, Hussein).
- Departments of Virology (Ghoniem), Animal Health Research Institute, Dokki, Egypt.
- Biotechnology (Aggour), Animal Health Research Institute, Dokki, Egypt.
- Department of Virology, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt (El Deeb, Hussein).
MeSH Terms
- Animals
- DNA Primers
- Herpesviridae Infections / diagnosis
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / genetics
- Herpesvirus 1, Equid / isolation & purification
- Herpesvirus 4, Equid / genetics
- Herpesvirus 4, Equid / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Influenza A virus / genetics
- Influenza A virus / isolation & purification
- Multiplex Polymerase Chain Reaction / methods
- Multiplex Polymerase Chain Reaction / veterinary
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / veterinary
- Orthomyxoviridae Infections / virology
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Respiratory Tract Diseases / diagnosis
- Respiratory Tract Diseases / veterinary
- Respiratory Tract Diseases / virology
- Sensitivity and Specificity
Conflict of Interest Statement
The authors declared no potential conflicts of interest with respect to the
research, authorship, and/or publication of this article.
References
This article includes 18 references
- Amer HM. Prevalence of equine herpes viruses 1, 2 and 4 in Arabian horse population in Egypt. Afr J Microbiol Res 2011;5:4805–4811.
- Ataseven VS, Dağalp SB, Güzel M, Başaran Z, Tan MT, Geraghty B. Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR.. Res Vet Sci 2009 Apr;86(2):339-44.
- Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG. Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).. Vet Microbiol 2007 Jul 20;123(1-3):93-103.
- Foord AJ, Selleck P, Colling A, Klippel J, Middleton D, Heine HG. Real-time RT-PCR for detection of equine influenza and evaluation using samples from horses infected with A/equine/Sydney/2007 (H3N8).. Vet Microbiol 2009 May 28;137(1-2):1-9.
- Gudnason H, Dufva M, Bang DD, Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature.. Nucleic Acids Res 2007;35(19):e127.
- Han JI, Chang DW, Na KJ. A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis.. J Vet Sci 2015;16(3):341-7.
- Hussein HA. Isolation and characterization of H3N8 equine influenza virus associated with 2008 EI outbreak in Egypt. Egypt J Virol 2011;8:1–12.
- Hussey SB, Clark R, Lunn KF, Breathnach C, Soboll G, Whalley JM, Lunn DP. Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.. J Vet Diagn Invest 2006 Jul;18(4):335-42.
- Ismail IM. An outbreak of equine influenza type (1) in Egypt in 1989. Vet Med J 1990;38:195–206.
- Jeggo MH. The initial laboratory diagnosis of equine influenza in Australia in 2007. Microbiol Aust 2008;29:80–82.
- Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF. Multiplex qPCR for detection and absolute quantification of malaria.. PLoS One 2013;8(8):e71539.
- Liss B. Improved quantitative real-time RT-PCR for expression profiling of individual cells.. Nucleic Acids Res 2002 Sep 1;30(17):e89.
- Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. 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 2009 Dec;47(12):3907-13.
- Pusterla N, Kass PH, Mapes S, Johnson C, Barnett DC, Vaala W, Gutierrez C, McDaniel R, Whitehead B, Manning J. Surveillance programme for important equine infectious respiratory pathogens in the USA.. Vet Rec 2011 Jul 2;169(1):12.
- Quinlivan M, Dempsey E, Ryan F, Arkins S, Cullinane A. Real-time reverse transcription PCR for detection and quantitative analysis of equine influenza virus.. J Clin Microbiol 2005 Oct;43(10):5055-7.
- Settypalli TB, Lamien CE, Spergser J, Lelenta M, Wade A, Gelaye E, Loitsch A, Minoungou G, Thiaucourt F, Diallo A. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.. PLoS One 2016;11(4):e0153688.
- Thonur L, Maley M, Gilray J, Crook T, Laming E, Turnbull D, Nath M, Willoughby K. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3.. BMC Vet Res 2012 Mar 28;8:37.
- Wernike K, Hoffmann B, Kalthoff D, König P, Beer M. Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1.. J Virol Methods 2011 Jun;174(1-2):77-84.
Citations
This article has been cited 3 times.- Altan E, Hui A, Li Y, Pesavento P, Asín J, Crossley B, Deng X, Uzal FA, Delwart E. New Parvoviruses and Picornavirus in Tissues and Feces of Foals with Interstitial Pneumonia. Viruses 2021 Aug 14;13(8).
- Jurisic L, Auerswald H, Marcacci M, Di Giallonardo F, Coetzee LM, Curini V, Averaimo D, Ortiz-Baez AS, Cammà C, Di Teodoro G, Richt JA, Holmes EC, Lorusso A. Insect-specific Alphamesonivirus-1 (Mesoniviridae) in lymph node and lung tissues from two horses with acute respiratory syndrome. J Virol 2025 Feb 25;99(2):e0214424.
- Sandybayev N, Strochkov V, Beloussov V, Orkara S, Kydyrmanov A, Khan Y, Batanova Z, Kassenov M. Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan. Vet World 2023 Aug;16(8):1682-1689.
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