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Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes

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Abstract

Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.

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Acknowledgments

The research project is implemented within the framework of the Action «Supporting Postdoctoral Researchers» of the Operational Program “Education and Lifelong Learning” (Action’s Beneficiary: General Secretariat for Research and Technology), and is co-financed by the European Social Fund (ESF) and the Greek State.

Author Contributions

D.K.T. conceived and designed the experiments; D.K.T and M.M. performed the experiments; D.K.T. and E.K. analyzed the data; D.K.T. wrote the paper; M.M. and E.K. proof-read the manuscript; D.K.T. and E.K. supervised the work.

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Authors and Affiliations

  1. Laboratory of Cellular Immunology, Department of Microbiology, Hellenic Pasteur Institute, 127 Vas. Sofias Ave., 11521, Athens, Greece

    Dimitra K. Toubanaki, Maritsa Margaroni & Evdokia Karagouni

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  1. Dimitra K. Toubanaki
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  2. Maritsa Margaroni
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  3. Evdokia Karagouni
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Correspondence to Dimitra K. Toubanaki or Evdokia Karagouni.

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The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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Toubanaki, D.K., Margaroni, M. & Karagouni, E. Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes. Curr Microbiol 71, 529–539 (2015). https://doi.org/10.1007/s00284-015-0880-0

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  • DOI: https://doi.org/10.1007/s00284-015-0880-0

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