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Research article - Peer-reviewed, 2010

Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene

Orozovic, Goran; Latorre-Margalef, Neus; Wahlgren, John; Muradrasoli, Shaman; Olsen, Björn


This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1-9). A total of 11 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny. (C) 2010 Elsevier B.V. All rights reserved.


Influenza A virus; Neuraminidase; Sequencing; PCR; Degenerate primers

Published in

Journal of Virological Methods
2010, volume: 170, number: 1-2, pages: 94-98

Authors' information

Orozovic, Goran
Latorre-Margalef, Neus
Wahlgren, John
Muradrasoli, Shaman
Swedish University of Agricultural Sciences, Department of Biomedical Science and Veterinary Public Health
Olsen, Björn
Uppsala University

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication Identifiers


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