title: High resolution melting analysis: rapid and precise characterisation of recombinant influenza A genomes.
abstract: BACKGROUND
  High resolution melting analysis (HRM) is a rapid and cost-effective technique for the characterisation of PCR amplicons. Because the reverse genetics of segmented influenza A viruses allows the generation of numerous influenza A virus reassortants within a short time, methods for the rapid selection of the correct recombinants are very useful.
  
  
  METHODS
  PCR primer pairs covering the single nucleotide polymorphism (SNP) positions of two different influenza A H5N1 strains were designed. Reassortants of the two different H5N1 isolates were used as a model to prove the suitability of HRM for the selection of the correct recombinants. Furthermore, two different cycler instruments were compared.
  
  
  RESULTS
  Both cycler instruments generated comparable average melting peaks, which allowed the easy identification and selection of the correct cloned segments or reassorted viruses.
  
  
  CONCLUSIONS
  HRM is a highly suitable method for the rapid and precise characterisation of cloned influenza A genomes.
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, Greifswald-Insel Riems, 17493, Germany. donata.kalthoff@fli.bund.de.
authors.forename: Donata
authors.initials: D
authors.lastname: Kalthoff
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
mesh_terms: D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D016865:Reassortant Viruses / Q000145:classification / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods; D044366:Transition Temperature; D014773:Virology / Q000379:methods*
chemical_list: D012367:RNA, Viral
references.citation: Clin Chem. 2003 Jun;49(6 Pt 1):853-60
references.pmid: 12765979
references.citation: Clin Chem. 2007 Jul;53(7):1377-80
references.pmid: 17525105
references.citation: Appl Environ Microbiol. 2005 Dec;71(12):8944-8
references.pmid: 16332896
references.citation: PLoS One. 2011;6(6):e21446
references.pmid: 21731753
references.citation: J Virol Methods. 2011 Jan;171(1):284-6
references.pmid: 20951745
references.citation: Pharmacogenomics. 2007 Jun;8(6):597-608
references.pmid: 17559349
references.citation: J Microbiol Methods. 2011 Sep;86(3):357-63
references.pmid: 21741999
references.citation: Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18590-5
references.pmid: 16339318
references.citation: J Clin Microbiol. 2008 Mar;46(3):1090-7
references.pmid: 18174299
references.citation: J Clin Microbiol. 2011 Oct;49(10):3555-9
references.pmid: 21865430
references.citation: J Mol Diagn. 2011 Nov;13(6):678-86
references.pmid: 21889610
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9345-50
references.pmid: 10430945
references.citation: Nucleic Acids Res. 2008 Dec;36(21):e139
references.pmid: 18832366
references.citation: J Clin Microbiol. 2011 Jul;49(7):2602-9
references.pmid: 21543559
references.citation: J Virol. 1999 Nov;73(11):9679-82
references.pmid: 10516084