2.5. Variation scores and host-specific amino acid residues in primer sequences
One of the concerns of designing primers targeting the AIV is unexpected point mutations, which could cause non-specific PCR amplification (i.e., false positives or negatives) and reduce sensitivity. Thus, the degree of variation at each nucleotide was further investigated; this process was conducted through the Analyze Sequence Variation (SNP) analysis of IRD using 80 H3N8 EIV and CIV sequences. In this approach, the least polymorphic (conserved) site was scored as 0, whereas the most variable site was marked as 200 (i.e., four nucleotides with 25% frequency each) ( Squires et al., 2012). Further, to characterize the properties of each primer sequence, annotations of the functional domains for the host-specific signatures were investigated through recent studies: Hu et al. (2014), Naffakh et al. (2008), and Chen et al. (2006).