Skip to main content

Advertisement

SpringerLink
Log in

Development of an updated PCR assay for detection of African swine fever virus

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

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
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Costard S, Mur L, Lubroth J, Sanchez-Vizcaino JM, Pfeiffer DU (2013) Epidemiology of African swine fever virus. Virus Res 173:191–197

    Article  CAS  PubMed  Google Scholar 

  2. Dixon LK, Chapman DA, Netherton CL, Upton C (2013) African swine fever virus replication and genomics. Virus Res 173:3–14

    Article  CAS  PubMed  Google Scholar 

  3. Denyer MS, Wilkinson PJ (1998) African swine fever. In: Encyclopedia of immunology, pp 54–56

  4. Rowlands RJ, Michaud V, Heath L, Hutchings G, Oura C, Vosloo W, Dwarka R, Onashvili T, Albina E, Dixon LK (2008) African swine fever virus isolate, Georgia, 2007. Emerg Infect Dis 14:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gogin A, Gerasimov V, Malogolovkin A, Kolbasov D (2013) African swine fever in the North Caucasus region and the Russian Federation in years 2007–2012. Virus Res 173:198–203

    Article  CAS  PubMed  Google Scholar 

  6. World Organization for Animal Health (OIE) (2014) World animal health information database (WAHID). http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Immsummary

  7. Sánchez-Vizcaíno JM, Mur L, Gomez-Villamandos JC, Carrasco L (2015) An update on the epidemiology and pathology of African swine fever. J Comp Pathol 152:9–21

    Article  PubMed  Google Scholar 

  8. Gallardo C, Reoyo A, Fernández-Pinero J, Iglesias I, Muñoz M, Arias M (2015) African swine fever: a global view of the current challenge. Porcine Health Manag 1:21

    Article  Google Scholar 

  9. Food and Agriculture Organization, United Nations (2013) African swine fever in the Russian Federation: risk factors for Europe and beyond. EMPRES WATCH, vol. 28. http://www.fao.org/docrep/018/aq240e/aq240e.pdf

  10. Oura CA, Edwards L, Batten CA (2013) Virological diagnosis of African swine fever—comparative study of available tests. Virus Res 173:150–158

    Article  CAS  PubMed  Google Scholar 

  11. Agüero M, Fernández J, Romero L, Sánchez Mascaraque C, Arias M, Sánchez-Vizcaíno JM (2003) Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. J Clin Microbiol 41:4431–4434

    Article  PubMed  PubMed Central  Google Scholar 

  12. King DP, Reid SM, Hutchings GH, Grierson SS, Wilkinson PJ, Dixon LK, Bastos AD, Drew TW (2003) Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods 107:53–61

    Article  CAS  PubMed  Google Scholar 

  13. Oura CA, Arias M (2012) African swine fever. In: OIE Biological Standards Commission (ed) OIE Manual of diagnostic tests and vaccines for terrestrial animals, 7th ed. Office International des Epizooties, Paris, France, pp 1069–1082

  14. Fernández-Pinero J, Gallardo C, Elizalde M, Robles A, Gómez C, Bishop R, Heath L, Couacy-Hymann E, Fasina FO, Pelayo V, Soler A, Arias M (2013) Molecular diagnosis of African swine fever by a new real-time PCR using universal probe library. Transbound Emerg Dis 60:48–58

    Article  PubMed  Google Scholar 

  15. Gallardo C, Nieto R, Soler A, Pelayo V, Fernández-Pinero J, Markowska-Daniel I, Pridotkas G, Nurmoja I, Granta R, Simón A, Pérez C, Martín E, Fernández-Pacheco P, Arias M (2015) Assessment of African swine fever diagnostic techniques as a response to the epidemic outbreaks in Eastern European Union countries: how to improve surveillance and control programs. J Clin Microbiol 53:2555–2565

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wilkinson PJ (2000) African swine fever. In: Manual of standards for diagnostic test and vaccines, 4th edn. Office International des Epizooties, Paris, France, pp 189–198

    Google Scholar 

  17. Zhao JJ, Cheng D, Li N, Sun Y, Shi Z, Zhu QH, Tu C, Tong GZ, Qiu HJ (2008) Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus. Vet Microbiol 126:1–10

    Article  CAS  PubMed  Google Scholar 

  18. Zhang XJ, Han QY, Sun Y, Zhang X, Qiu HJ (2012) Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1. Res Vet Sci 92:512–518

    Article  CAS  PubMed  Google Scholar 

  19. Haines FJ, Hofmann MA, King DP, Drew TW, Crooke HR (2013) Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses. PLoS One 8:e71019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Steiger Y, Ackermann M, Mettraux C, Kihm U (1992) Rapid and biologically safe diagnosis of African swine fever virus infection by using polymerase chain reaction. J Clin Microbiol 30:1–8

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Mo Zhou at Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, for revising the manuscript.

Author information

Author notes

    Authors and Affiliations

    1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute (HVRI), Chinese Academy of Agricultural Sciences, 678 Haping Road, Harbin, 150069, China

      Yuzi Luo, Lina Shao, Yuan Sun, Yan Liu, Shengwei Ji, Xing-Yu Meng, Su Li, Yongfeng Li & Hua-Ji Qiu

    2. Department of Microbiology, National Veterinary Institute (SVA), 75189, Uppsala, Sweden

      Frederik Widén & Lihong Liu

    3. Department for Disease Control and Epidemiology, National Veterinary Institute (SVA), 75189, Uppsala, Sweden

      Karl Ståhl

    4. National Animal Disease Diagnostics and Epidemiology Center (NADDEC), Ministry of Agricultural, Animal Industry and Fisheries, Entebbe, Uganda

      Stella A. Atim & Chrisostom Ayebazibwe

    5. Department of Biological Sciences, College of Natural Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda

      Charles Masembe

    6. Joint Laboratory on Veterinary Microbiology between HVRI and SVA, 678 Haping Road, Harbin, 150069, China

      Yuzi Luo, Lina Shao, Yuan Sun, Yan Liu, Shengwei Ji, Xing-Yu Meng, Su Li, Yongfeng Li, Karl Ståhl, Frederik Widén, Lihong Liu & Hua-Ji Qiu

    Authors
    1. Yuzi Luo
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Stella A. Atim
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Lina Shao
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Chrisostom Ayebazibwe
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Yuan Sun
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Yan Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Shengwei Ji
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Xing-Yu Meng
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Su Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Yongfeng Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Charles Masembe
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. Karl Ståhl
      View author publications

      You can also search for this author in PubMed Google Scholar

    13. Frederik Widén
      View author publications

      You can also search for this author in PubMed Google Scholar

    14. Lihong Liu
      View author publications

      You can also search for this author in PubMed Google Scholar

    15. Hua-Ji Qiu
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Hua-Ji Qiu.

    Ethics declarations

    Funding

    This work was supported by grants from the European Union’s Seventh Framework Programme LinkTADs (No. 613804), the Harbin Animal Husbandry and Veterinary Bureau of China, and the OIE twinning project between SVA, Sweden and MAAIF-NADDEC, Uganda. Charles Masembe was funded by the Wellcome Trust Fellowship.

    Conflict of interest

    All authors declare that they have no competing interests.

    Ethical approval

    None of the experiments in this study involved human participants.

    Additional information

    Y. Luo, S. A. Atim and L. Shao contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Luo, Y., Atim, S.A., Shao, L. et al. Development of an updated PCR assay for detection of African swine fever virus. Arch Virol 162, 191–199 (2017). https://doi.org/10.1007/s00705-016-3069-3

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00705-016-3069-3

    Keywords

    Search

    Navigation