Skip to main content
SpringerLink
Log in

Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study

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

Abstract

Classical swine fever (CSF) is a devastating animal disease of great economic impact worldwide. In many countries, CSF has been endemic for decades, and vaccination of domestic pigs is one of the measures to control the disease. Consequently, differentiating infected from vaccinated animals by antibody ELISA screening is not applicable. In some countries, such as Cuba, lack of molecular techniques for sensitive, rapid and reliable detection of virus genomes is a critical point. To overcome this problem, an easy-to-use one-tube assay based on the loop-mediated isothermal amplification (LAMP) principle has been developed for detection of the genome of CSF virus (CSFV) of endemic Cuban genotype 1.4 isolates. The assay reliably detected recent isolates from three different regions of Cuba with an analytical sensitivity 10-100 times lower than that of quantitative reverse transcription RT-qPCR. Diagnostic test sensitivity was examined using reference sera from two groups of pigs experimentally infected with Cuban virulent strain CSF0705 “Margarita” and the recent field isolate CSF1058 “Pinar del Rio”. Differences in pathogenicity of the two viruses were reflected in the clinical course of disease as well as in virus loads of blood samples. Low viral RNA loads in samples from pigs infected with the field isolate caused serious detection problems in RT-LAMP as well as in RT-qPCR. Thus, it will be necessary in future research to focus on targeted sampling of diseased animals and to restrict diagnosis to the herd level in order to establish LAMP as an efficient tool for diagnosing CSF under field conditions.

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. Moennig V, Floegel-Niesmann G, Greiser-Wilke I (2003) Clinical signs and epidemiology of classical swine fever: a review of new knowledge. Vet J 165(1):11–20

    Article  CAS  PubMed  Google Scholar 

  2. Edwards S, Fukusho A, Lefevre PC, Lipowski A, Pejsak Z, Roehe P, Westergaard J (2000) Classical swine fever: the global situation. Vet Microbiol 73(2–3):103–119

    Article  CAS  PubMed  Google Scholar 

  3. Pereda AJ, Greiser-Wilke I, Schmitt B, Rincon MA, Mogollon JD, Sabogal ZY, Lora AM, Sanguinetti H, Piccone ME (2005) Phylogenetic analysis of classical swine fever virus (CSFV) field isolates from outbreaks in South and Central America. Virus Res 110(1–2):111–118

    Article  CAS  PubMed  Google Scholar 

  4. Sabogal ZY, Mogollon JD, Rincon MA, Clavijo A (2006) Phylogenetic analysis of recent isolates of classical swine fever virus from Colombia. Virus Res 115(1):99–103

    Article  CAS  PubMed  Google Scholar 

  5. Arainga M, Hisanaga T, Hills K, Handel K, Rivera H, Pasick J (2010) Phylogenetic analysis of classical swine fever virus isolates from Peru. Transbound Emerg Dis 57(4):262–270

    CAS  PubMed  Google Scholar 

  6. OIE (2013) WAHID home page. http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Diseasetimelines

  7. Paton DJ, McGoldrick A, Greiser-Wilke I, Parchariyanon S, Song JY, Liou PP, Stadejek T, Lowings JP, Bjorklund H, Belak S (2000) Genetic typing of classical swine fever virus. Vet Microbiol 73(2–3):137–157

    Article  CAS  PubMed  Google Scholar 

  8. Postel A, Schmeiser S, Perera CL, Rodriguez LJ, Frias-Lepoureau MT, Becher P (2013) Classical swine fever virus isolates from Cuba form a new subgenotype 1.4. Vet Microbiol 161(3–4):334–338

    Article  CAS  PubMed  Google Scholar 

  9. de Arce HD, Ganges L, Barrera M, Naranjo D, Sobrino F, Frias MT, Nunez JI (2005) Origin and evolution of viruses causing classical swine fever in Cuba. Virus Res 112(1–2):123–131

    Article  PubMed  Google Scholar 

  10. de Arce HD, Nunez JI, Ganges L, Barreras M, Teresa Frias M, Sobrino F (1999) Molecular epidemiology of classical swine fever in Cuba. Virus Res 64(1):61–67

    Article  Google Scholar 

  11. Perez LJ, Diaz de Arce H, Perera CL, Rosell R, Frias MT, Percedo MI, Tarradas J, Dominguez P, Nunez JI, Ganges L (2012) Positive selection pressure on the B/C domains of the E2-gene of classical swine fever virus in endemic areas under C-strain vaccination. Infect Genet Evol 12(7):1405–1412

    Article  PubMed  Google Scholar 

  12. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Chen HT, Zhang J, Ma LN, Ma YP, Ding YZ, Liu XT, Chen L, Ma LQ, Zhang YG, Liu YS (2009) Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification. Mol Cell Probes 23(2):71–74

    Article  PubMed  Google Scholar 

  14. Chen L, Fan XZ, Wang Q, Xu L, Zhao QZ, Zhou YC, Liu J, Tang B, Zou XQ (2010) A novel RT-LAMP assay for rapid and simple detection of classical swine fever virus. Virol Sin 25(1):59–64

    Article  PubMed  Google Scholar 

  15. Chowdry VK, Luo Y, Widen F, Qiu HJ, Shan H, Belak S, Liu L (2014) Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field. J Virol Methods 197:14–18

    Article  CAS  PubMed  Google Scholar 

  16. Yin S, Shang Y, Zhou G, Tian H, Liu Y, Cai X, Liu X (2010) Development and evaluation of rapid detection of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP). J Biotechnol 146(4):147–150

    Article  CAS  PubMed  Google Scholar 

  17. Zhang XJ, Han QY, Sun Y, Belak S, Liu L, Qiu HJ (2011) Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China. J Virol Methods 171(1):200–205

    Article  CAS  PubMed  Google Scholar 

  18. Zhang XJ, Sun Y, Liu L, Belak S, Qiu HJ (2010) Validation of a loop-mediated isothermal amplification assay for visualised detection of wild-type classical swine fever virus. J Virol Methods 167(1):74–78

    Article  CAS  PubMed  Google Scholar 

  19. Hoffmann B, Beer M, Schelp C, Schirrmeier H, Depner K (2005) Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever. J Virol Methods 130(1–2):36–44

    Article  CAS  PubMed  Google Scholar 

  20. The Classical Swine Fever Database—Version 3 (2013) EU and OIE Reference Laboratory for Classical Swine Fever. http://viro08.tiho-hannover.de/eg/csf/

  21. Mittelholzer C, Moser C, Tratschin JD, Hofmann MA (2000) Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet Microbiol 74(4):293–308

    Article  CAS  Google Scholar 

  22. Anonymous (2007) EU Diagnostic Manual for Classical swine fever (CSF) diagnosis: Technical part. EU and OIE Reference Laboratory for CSF, Homepage. http://viro08.tiho-hannover.de/eg/Technical_Annex_Draft_2007.pdf. Accessed 1 June 2014

  23. Postel A, Letzel T, Frischmann S, Grund C, Beer M, Harder T (2010) Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes. J Vet Diagn Invest 22(1):61–66

    Article  PubMed  Google Scholar 

  24. Tarradas J, Argilaguet JM, Rosell R, Nofrarias M, Crisci E, Cordoba L, Perez-Martin E, Diaz I, Rodriguez F, Domingo M, Montoya M, Ganges L (2010) Interferon-gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs. Vet Microbiol 142(1–2):51–58

    Article  CAS  PubMed  Google Scholar 

  25. Ganges L, Barrera M, Diaz de Arce H, Vega A, Nunez JI, Sobrino F, Frias MT (2007) Antigenic, biological and molecular characterization of the Cuban CSFV isolate “Margarita”. Rev Salud Anim 29(3):182–192

    Google Scholar 

  26. Sanchez O, Barrera M, Rodriguez MP, Frias MT, Figueroa NE, Naranjo P, Montesino R, Farnos O, Castell S, Venereo A, Ganges L, Borroto C, Toledo JR (2008) Classical swine fever virus E2 glycoprotein antigen produced in adenovirally transduced PK-15 cells confers complete protection in pigs upon viral challenge. Vaccine 26(7):988–997

    Article  CAS  PubMed  Google Scholar 

  27. Barrera M, Sanchez O, Farnos O, Rodriguez MP, Dominguez P, Tait H, Frias M, Avila M, Vega E, Toledo JR (2010) Early onset and long lasting protection in pigs provided by a classical swine fever E2-vaccine candidate produced in the milk of goats. Vet Immunol Immunopathol 133(1):25–32

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study is dedicated to our deceased colleague Irene Greiser-Wilke. She initiated the OIE twinning project between CENSA, Cuba, and the EU and OIE RL for CSF, Germany, and had the vision to implement a LAMP assay for CSF diagnosis in Cuba. The study was financed by the OIE in the framework of the twinning project “Support to the CENSA Laboratory for CSF for the Planned Establishment of an OIE Reference Laboratory” and by the Institute of Virology, University of Veterinary Medicine in Hannover. We wish to thank the entire staff of the EU and OIE RL for CSF. We are grateful to have with Monika Berg, Holger Mosch and Günter Thiem an outstanding animal care team. We thank Benjamin Ostermann for his support in the initial phase of the study, and Inga Grotha for her tireless efforts and excellent technical assistance.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. EU and OIE Reference Laboratory for Classical Swine Fever, Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559, Hannover, Germany

    Alexander Postel, Stefanie Schmeiser, Denise Meyer, Alexandra Meindl-Boehmer, Sophia Austermann-Busch & Paul Becher

  2. Centro Nacional de Sanidad Agropecuaria (CENSA), San Jose de las Lajas, Apdo. 10, Mayabeque, Cuba

    Lester J. Pérez, Carmen L. Perera, Liliam Rios & Maria T. Frias-Lepoureau

Authors
  1. Alexander Postel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lester J. Pérez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carmen L. Perera
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefanie Schmeiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Denise Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexandra Meindl-Boehmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liliam Rios
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sophia Austermann-Busch
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Maria T. Frias-Lepoureau
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Paul Becher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul Becher.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Postel, A., Pérez, L.J., Perera, C.L. et al. Development of a new LAMP assay for the detection of CSFV strains from Cuba: a proof-of-concept study. Arch Virol 160, 1435–1448 (2015). https://doi.org/10.1007/s00705-015-2407-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-015-2407-1

Keywords

Search

Navigation