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

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 pathogenicit...

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Bibliographic Details
Published in:Current microbiology 2015-11, Vol.71 (5), p.529-539
Main Authors: Toubanaki, Dimitra K., Margaroni, Maritsa, Karagouni, Evdokia
Format: Article
Language:English
Subjects:
Alleles
Animals
Aquaculture
Biomedical and Life Sciences
Biotechnology
Economic impact
Fish Diseases - virology
Genetic diversity
Genotype
Genotype & phenotype
Genotypes
Life Sciences
Microbiology
Molecular Typing - methods
Nodaviridae - classification
Nodaviridae - genetics
Nodavirus
Pathogens
Polymerase chain reaction
Polymerase Chain Reaction - methods
Reagents
Sensitivity and Specificity
Viruses
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Summary: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.
ISSN:0343-8651
1432-0991
DOI:10.1007/s00284-015-0880-0