Advanced

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

Orozovic, Goran; Latorre-Margalef, Neus LU ; Wahlgren, John; Muradrasoli, Shaman and Olsen, Björn (2010) In Journal of Virological Methods 170(1-2). p.94-98
Abstract

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 115 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... (More)

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 115 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.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Degenerate primers, Influenza A virus, Neuraminidase, PCR, Sequencing
in
Journal of Virological Methods
volume
170
issue
1-2
pages
5 pages
publisher
Elsevier
external identifiers
  • scopus:78249257293
ISSN
0166-0934
DOI
10.1016/j.jviromet.2010.09.006
language
English
LU publication?
no
id
b57f4b68-8af6-4a91-b664-2cd03c08e08e
date added to LUP
2017-04-11 14:06:00
date last changed
2018-06-17 05:22:12
@article{b57f4b68-8af6-4a91-b664-2cd03c08e08e,
  abstract     = {<p>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 115 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.</p>},
  author       = {Orozovic, Goran and Latorre-Margalef, Neus and Wahlgren, John and Muradrasoli, Shaman and Olsen, Björn},
  issn         = {0166-0934},
  keyword      = {Degenerate primers,Influenza A virus,Neuraminidase,PCR,Sequencing},
  language     = {eng},
  number       = {1-2},
  pages        = {94--98},
  publisher    = {Elsevier},
  series       = {Journal of Virological Methods},
  title        = {Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene},
  url          = {http://dx.doi.org/10.1016/j.jviromet.2010.09.006},
  volume       = {170},
  year         = {2010},
}