High resolution melting analysis: rapid and precise characterisation of recombinant influenza A genomes

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.