Standarization of a SYBR Green-I based real time RT-PCR assay for the detection of equine influenza virus

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Published: Jan 1, 2018
Keywords:
equine influenza virus, SYBR Green-I based real time RT-PCR assay

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Ana María Acevedo
National Center for Animal and Plant Health
Ana María Lazo
National Center for Animal and Plant Health
Damarys Relova
National Center for Animal and Plant Health
Carmen Laura Perera
National Center for Animal and Plant Health

Abstract

Equine influenza is an acute respiratory infection of horses, donkeys, mules and zebras. Nowadays, the reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR (rRT-PCR) assays are being widely used in diagnostic laboratories as a more sensitive alternative to virus isolation for the detection of equine influenza virus. The purpose of this study was to standardize an rRT-PCR, based on SYBR Green-I coupled with melting curve analysis for the detection of equine influenza virus (gene M) and the typing of hemagglutinin gene (HA). Sensitivity and specificity of the assay (gene M) were evaluated. In terms of the matrix gene copy number, the detection limit was 4.5 gene copies/μl of in vitro transcribed RNA. The linear range obtained for the transcript in the assay genered a typical standard curve from 107 until 100 gene copies/µl in terms of RNA copy number. The correlation coefficient (R2) was 0.99 for the RNA copies detected in nuclease free water and the error of standard curve was 0.0139. The rRT-PCR assay showed to be specific for equine influenza virus. No specific amplification curves were obtained with any of the other viruses tested such as equine viral arteritis, equine herpesvirus-1 and equine herpesvirus-4. The assay showed amplification and specific melting curves when the second set of primers against HA gene was evaluated. Thus, the proposed SYBR Green-I based rRT-PCR assay for the detection of equine influenza virus can be used in case of a possible emergency of this agent in Cuba.

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How to Cite
1.
Acevedo AM, Lazo AM, Relova D, Perera CL. Standarization of a SYBR Green-I based real time RT-PCR assay for the detection of equine influenza virus. Rev. Salud Anim. [Internet]. 2018 Jan. 1 [cited 2024 Nov. 22];40(3). Available from: https://revistas.censa.edu.cu/index.php/RSA/article/view/996
Issue
Vol. 40 No. 3 (2018): septiembre-diciembre
Section
ARTÍCULOS ORIGINALES
National Center for Animal and Plant Health (CENSA)

References

Sovinova´O, Tu¨mova´B, Pouska F, Nemec J. Isolation of a virus causing respiratory disease in horses. Acta Virol. 1958;2:52-61.

Wadell GH, Teigland MB, Siegel MM. A new in?uenza virus associated with equine respiratory disease. J Am Vet Med Assoc. 1963;143:587-590.

Webster RG. Are equine 1 influenza viruses still present in horses? Equine Vet J. 1993;25: 537-538.

Office International des Epizooties. Equine influenza. Manual of standards for diagnostic tests and vaccines. Office International des Epizooties, Paris, France. 2000:546-557.

Office International des Epizooties. Conclusions and recommendations from the consultation meeting of OIE and W.H.O. experts on equine influenza, Newmarket, United Kingdom, 18-19 September 1995. Office Int. Epizoot. Bull. 1996;108:482-484

Morley PS, Bogdan JR, Townsend HG, Haines DM. Evaluation of Directigen Flu A assay for detection of influenza antigen in nasal secretions of horses. Equine Vet J. 1995;27:131-134.

Quinlivan MA, Cullinane M, Nelly K, Van Maanen, Heldens J, Arkins S. Comparison of sensitivities of virus isolation, antigen detection,and nucleic acid amplification for detection of equine influenza virus. J Clin Microbiol. 2004;42:759-763.

Foord AJ, Selleck P, Colling A, Klippel J, Middleton D, Heine H. 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;137:1-9.

Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ,et al. 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;47:3907-3913.

Aeschbacher S, Santschi E, Gerber V, Stalder HP, Zanoni RG. Development of a real-time RT-PCR for detection of equine influenza virus. Schweiz Arch Tierheilkd. 2015;157(4):191-201.

Balasuriya Udeni BR, Lee Pei-Yu, Tiwari A, Skillman A, Nam B, Chambers TM, et al. Rapid detection of equine influenza virus H3N8 subtype by insulatedisothermal RT-PCR (iiRT-PCR) assay using the POCKITTMNucleic Acid Analyzer. J Virol Methods. 2014;207:66-72.

Rios L, Perera CL, Coronado L, Relova D, Álvarez AM, Ganges L, Díaz de Arce H, Núñez JI, Pérez LJ. Strategy for Pan/Foot-and-Mouth Disease Virus (FMDV) Detection: A Combination of Sequences Analysis, in Silico Predictions and Laboratory Diagnostic Evaluation. Front Vet Sci. 2018;5:1-13.

Chen J, Zhao Z, Chen Y, Zhang J, Yan L, Zheng X, et al. Development and application of a SYBR green real-time PCR for detection of the emerging avian leukosis virus subgroup K. Poult Sci. 2018;97(7):2568-2574.

Chen GH, Tang XY, Sun Y, Zhou L, Li D, Bai Y, et al. Development of a SYBR Green-based real-time quantitative PCR assay to detect PCV3 in pigs. J Virol Methods. 2018;251:129-132.

Zhu Z, Fan H, Qi X, Qi Y, Shi Z, Wang H,et al. Development and evaluation of a SYBR Green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9) virus. PLoS One. 2013;8(11):e80028.

Acevedo AM, Perera CL, Vega A, Ríos L, Coronado L, Relova D, et al. A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus. Mol Cell Probe. 2013;27:184-192.

Prieto A, Díaz-Cao JM, Fernández AR, Panadero R, Díaz P, López C, et al. (2014). Application of real-time PCR to detect Aleutian Mink Disease Virus on environmental farm sources. Vet Microbiol. 173;355-359.

Yang Y, Qin X, Zhang W, Li Y, Zhang Z. Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays. Mol Cell Probe. 2016;30:300-305.

Wang J, Liu L, Li R, Wang J, Fu Q, Yuan W. Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification. Arch. Virol. 2016;161:1015-1018.

Díaz de Arce H, Pérez LJ, Frías MT, Rosell R, Tarradas J, Núñez JI, et al. A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections. Vet Microbiol. 2009;139:245-252.

Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 haemagglutinin subtypes. J Clin Microbiol. 2002;40:3256-3260.

Navarro E, Serrano-Heras G, Castaño MJ, Solera J. Real-time PCR detection chemistry. Clin Chim Acta. 2015;439:231-250.

Wernike K, Beer M, Hoffmann B. Rapid detection of foot-and-mouth disease virus, influenza A virus and classical swine fever virus by high-speed real-time RT-PCR. J Virol Methods. 2013;193(1):50-54.

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;43: 5055-5057.

Website http://www.qiagen.com. Application Note. Comparing SYBR(r) Green- and probe-based detection in real-time PCR. QIAGEN. 2017.

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