126.96.36.199. What do we want to detect?
The first decision is to establish precisely what the assay should detect and what it should not detect:
- For distinguishing closely related strains, locating the 3’ terminus of one primer at a position where the strains differ consistently. The other primer can be common for all strains (de Miranda et al., 2010b).
- For detecting all potential variants within a virus species or complex, the primer sequences should be conserved between all known variants, so as to be able to detect both known and as-yet-unknown variants in the complex. Locate the primers at least 200 nucleotides apart, so that new variants can be identified by sequence analyses of the intervening region.
- Avoid locating the 3’ terminus of a primer on the 3rd base of a codon in the coding region of a virus genome, since these are by far the most variable nucleotides in any virus genome (Grabensteiner et al., 2001; Bakonyi et al., 2002b; de Miranda et al., 2004; Lanzi et al., 2006; Olivier et al., 2008; de Miranda et al., 2010b).
- Use deoxyinosine as the 3’
nucleotide, which can pair with all nucleotides (Benjeddou et al., 2001;
Topley et al., 2005), thus avoiding the 3’ mismatch problem altogether.