Table 1 Selected parameter settings for round one in the primer design process.

Primer length

18 – 22 nucleotides

Product length

80 – 120 nucleotides

Primer melting temperature

58.0 – 60.0 °

Max. primer melting temperature diff.

2.0 °

Enforce G/C terminal

Yes

G/C content

40.0 – 60.0%

Max. base repeat

2 (e.g., GGG in a primer is disallowed)

Force specificity for both primers

Yes (i.e., none of the two primers has a perfect alignment to a non-target)