Skip to main content
SpringerLink
Log in

Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50–1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Allen GP, Bryans JT (1986) Molecular epizootiology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections. Prog Vet Microbiol Immunol 2:78–144

    CAS  PubMed  Google Scholar 

  • Allen GP, Kydd JH, Slater JD, Smith KC (2004) Equid herpesvirus 1 and equid herpesvirus 4 infections. In: Coetzer JAW, Tustin (eds) Infectious diseases of livestock. Oxford University, Oxford, pp 829–859

  • Ataseven VS, Dagalp SB, Guzel M, Basaran Z, Tan MT, Geraghty B (2009) 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 86(2):339–344

    Article  CAS  PubMed  Google Scholar 

  • Bagust TJ (1972) A review of viral infections of horses. Aust Vet J 48(9):520–523

    Article  CAS  PubMed  Google Scholar 

  • Carvalho R, Passos LM, Martins AS (2000) Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool. J Vet Med B Infect Dis Vet Public Health 47(5):351–359

    Article  CAS  PubMed  Google Scholar 

  • Cheng D, Zhao JJ, Li N, Sun Y, Zhou YJ, Zhu Y, Tian ZJ, Tu C, Tong GZ, Qiu HJ (2008) Simultaneous detection of classical swine fever virus and North American genotype porcine reproductive and respiratory syndrome virus using a duplex real-time RT-PCR. J Virol Methods 151(2):194–199

    Article  CAS  PubMed  Google Scholar 

  • Cheng J, Jiang Y, Rao P, Wu H, Dong Q, Wu Z, Ding X, Guo J (2013) Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen. Arch Virol 158(2):379–386

    Article  CAS  PubMed  Google Scholar 

  • Crabb BS, Studdert MJ (1993) Epitopes of glycoprotein G of equine herpesviruses 4 and 1 located near the C termini elicit type-specific antibody responses in the natural host. J Virol 67(10):6332–6338

    CAS  PubMed Central  PubMed  Google Scholar 

  • Diallo IS, Hewitson G, Wright L, Rodwell BJ, Corney BG (2006) Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction. J Virol Methods 131(1):92–98

    Article  CAS  PubMed  Google Scholar 

  • Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG (2007) Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). Vet Microbiol 123(1–3):93–103

    Article  CAS  PubMed  Google Scholar 

  • Eischeid AC (2011) SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR. BMC Res Notes 4:263

    Article  PubMed Central  PubMed  Google Scholar 

  • Elbir H, Henry M, Diatta G, Mediannikov O, Sokhna C, Tall A, Socolovschi C, Cutler SJ, Bilcha KD, Ali J, Campelo D, Barker SC, Raoult D, Drancourt M (2013) Multiplex real-time PCR diagnostic of relapsing fevers in Africa. PLoS Negl Trop Dis 7(1):e2042

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Foote CE, Love DN, Gilkerson JR, Whalley JM (2004) Detection of EHV-1 and EHV-4 DNA in unweaned thoroughbred foals from vaccinated mares on a large stud farm. Equine Vet J 36(4):341–345

    Article  CAS  PubMed  Google Scholar 

  • Hartley WJ, Dixon RJ (1979) An outbreak of foal perinatal mortality due to equid herpesvirus type 1: pathological observations. Equine Vet J 11(4):215–218

    Article  CAS  PubMed  Google Scholar 

  • Hebia-Fellah I, Leaute A, Fieni F, Zientara S, Imbert-Marcille BM, Besse B, Fortier G, Pronost S, Miszczak F, Ferry B, Thorin C, Pellerin JL, Bruyas JF (2009) Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR). Theriogenology 71(9):1381–1389

    Article  CAS  PubMed  Google Scholar 

  • Hernandez-Arteaga S, Lopez-Revilla R (2008) Quantitation of human papillomavirus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen. Anal Biochem 380(1):131–133

    Article  CAS  PubMed  Google Scholar 

  • Ihrig J, Lill R, Muhlenhoff U (2006) Application of the DNA-specific dye EvaGreen for the routine quantification of DNA in microplates. Anal Biochem 359(2):265–267

    Article  CAS  PubMed  Google Scholar 

  • Khan SA, Sung K, Nawaz MS (2011) Detection of aacA-aphD, qacEdelta1, marA, floR, and tetA genes from multidrug-resistant bacteria: comparative analysis of real-time multiplex PCR assays using EvaGreen((R)) and SYBR((R)) Green I dyes. Mol Cell Probes 25(2–3):78–86

    Article  CAS  PubMed  Google Scholar 

  • Kirisawa R, Endo A, Iwai H, Kawakami Y (1993) Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction. Vet Microbiol 36(1–2):57–67

    Article  CAS  PubMed  Google Scholar 

  • Lawrence GL, Gilkerson J, Love DN, Sabine M, Whalley JM (1994) Rapid, single-step differentiation of equid herpesviruses 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers. J Virol Methods 47(1–2):59–72

    Article  CAS  PubMed  Google Scholar 

  • Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY (2010) A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants. J Integr Plant Biol 52(12):1036–1042

    Article  CAS  PubMed  Google Scholar 

  • Mao F, Leung WY, Xin X (2007) Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 7:76

    Article  PubMed Central  PubMed  Google Scholar 

  • Monti M, Martini M, Tedeschi R (2013) EvaGreen real-time PCR protocol for specific 'Candidatus Phytoplasma mali' detection and quantification in insects. Mol Cell Probes 27(3–4):129–136

    Article  CAS  PubMed  Google Scholar 

  • Mossbrugger I, Felder E, Gramsamer B, Wolfel R (2013) EvaGreen based real-time RT-PCR assay for broad-range detection of hantaviruses in the field. J Clin Virol 58(1):334–335

    Article  PubMed  Google Scholar 

  • Patel JR, Heldens J (2005) Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)—epidemiology, disease and immunoprophylaxis: a brief review. Vet J 170(1):14–23

    Article  CAS  PubMed  Google Scholar 

  • Perkins GA, Goodman LB, Dubovi EJ, Kim SG, Osterrieder N (2008) Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR. J Vet Intern Med 22(5):1234–1238

    Article  CAS  PubMed  Google Scholar 

  • Pusterla N, Wilson WD, Mapes S, Finno C, Isbell D, Arthur RM, Ferraro GL (2009) Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California. Vet J 179(2):230–239

    Article  CAS  PubMed  Google Scholar 

  • Pusterla N, Mapes S, David Wilson W (2012) Prevalence of latent alpha-herpesviruses in thoroughbred racing horses. Vet J 193(2):579–582

    Article  CAS  PubMed  Google Scholar 

  • Safdar M, Abasiyanik MF (2013) Simultaneous identification of pork and poultry origins in pet foods by a quick multiplex real-time PCR assay using EvaGreen florescence dye. Appl Biochem Biotechnol. doi:10.1007/s12010-013-0485-7

  • Sang F, Ren J (2006) Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen. J Sep Sci 29(9):1275–1280

    Article  CAS  PubMed  Google Scholar 

  • Sharma PC, Cullinane AA, Onions DE, Nicolson L (1992) Diagnosis of equid herpesviruses-1 and -4 by polymerase chain reaction. Equine Vet J 24(1):20–25

    Article  CAS  PubMed  Google Scholar 

  • Slater J (2007) Equine herpesviruses. In: Equine infectious disease. WB Saunders, Saint Louis, USA, pp 134–153

    Book  Google Scholar 

  • Studdert MJ, Hartley CA, Dynon K, Sandy JR, Slocombe RF, Charles JA, Milne ME, Clarke AF, El-Hage C (2003) Outbreak of equine herpesvirus type 1 myeloencephalitis: new insights from virus identification by PCR and the application of an EHV-1-specific antibody detection ELISA. Vet Rec 153(14):417–423

    Article  CAS  PubMed  Google Scholar 

  • van Maanen C (2002) Equine herpesvirus 1 and 4 infections: an update. Vet Q 24(2):58–78

    PubMed  Google Scholar 

  • van Maanen C, de Boer-Luijtze E, Terpstra C (2000) Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4). Vet Microbiol 71(1–2):37–51

    Article  PubMed  Google Scholar 

  • Wang W, Chen K, Xu C (2006) DNA quantification using EvaGreen and a real-time PCR instrument. Anal Biochem 356(2):303–305

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Raidal SL, Pizzirani A, Wilcox GE (2007) Detection of respiratory herpesviruses in foals and adult horses determined by nested multiplex PCR. Vet Microbiol 121(1–2):18–28

    Article  CAS  PubMed  Google Scholar 

  • Whelan JA, Russell NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278(1–2):261–269

    Article  CAS  PubMed  Google Scholar 

  • Zhang N, Fang S, Wang T, Li J, Cheng X, Zhao C, Wang X, Lv X, Wu C, Zhang R, Cheng J, Xue H, Lu Z (2012a) Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens. Appl Microbiol Biotechnol 93(2):797–805

    Article  CAS  PubMed  Google Scholar 

  • Zhang P, Liu Y, Alsarakibi M, Li J, Liu T, Li Y, Li G (2012b) Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 111(5):2157–2163

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Zhige Tian for her experimental assistance. This study was supported by grants from the Central Public-interest Scientific Institution Basal Research Fund (0302012011), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAD46B01-02 & 2012BAD46B03), and Special Fund for Agro-scientific Research in the Public Interest (201003075).

Conflict of interest

The authors have no conflicts of interest.

Author information

Authors and Affiliations

  1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, 427 Maduan Street, Harbin, 150001, People’s Republic of China

    Zhe Hu, Chao Zhu, Hao Chang, Wei Guo, Diqiu Liu, Wenhua Xiang & Xiaojun Wang

Authors
  1. Zhe Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chao Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hao Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Diqiu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenhua Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaojun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaojun Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, Z., Zhu, C., Chang, H. et al. Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4. Appl Microbiol Biotechnol 98, 4179–4186 (2014). https://doi.org/10.1007/s00253-014-5626-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5626-6

Keywords

Search

Navigation