Archives of Virology

, Volume 157, Issue 5, pp 833–844 | Cite as

Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus

  • Alia Yacoub
  • Mikael Leijon
  • Michael J. McMenamy
  • Karin Ullman
  • John McKillen
  • Gordon Allan
  • Sándor Belák
Original Article


A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.


Newcastle Disease Virus Avian Influenza Virus Newcastle Disease Infectious Bronchitis Virus TaqMan Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the Award of Excellence of the Swedish University of Agricultural Sciences (2007) granted to SB, the EU grant FLUTEST (SSPE-CT-2007-044429), a grant from the Swedish Civil Contingencies Agency (2:4 Krisberedskap) and a grant in the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission – Directorate General Home Affairs. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Dr. Béla Lomniczi (VMRI, Budapest, Hungary) is thanked for kindly providing many NDV isolates used in developing this assay and for constructive criticism and advice. We thank Drs. István Kiss and Siamak Zohari for useful discussions. Ulla Berglöf is thanked for expert technical assistance. The critical reading and suggestions of Dr. László Zsák (USDA; ARS, Southeast Poultry Research Laboratory, Athens, GA, USA) are highly appreciated.


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Alia Yacoub
    • 1
    • 5
  • Mikael Leijon
    • 1
    • 5
  • Michael J. McMenamy
    • 2
  • Karin Ullman
    • 1
  • John McKillen
    • 3
  • Gordon Allan
    • 2
  • Sándor Belák
    • 1
    • 4
    • 5
  1. 1.Department of Virology, Immunobiology and ParasitologyNational Veterinary Institute (SVA)UppsalaSweden
  2. 2.School of Biological SciencesQueen’s University BelfastBelfastUK
  3. 3.Veterinary Sciences DivisionAgri-Food and Biosciences InstituteBelfastUK
  4. 4.Department of Biomedical Sciences and Veterinary Public HealthSwedish University of Agricultural Sciences (SLU)UppsalaSweden
  5. 5.The Joint R&D Division in Virology of SVA and SLUThe OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary MedicineUppsalaSweden

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