Primer Design

Primers for the target sequences were designed using Primer3Plus (version 0.4.2). The primer parameters were configured with an optimal melting temperature of 60℃ (ranging from 58℃ to 62℃)), an optimal primer length of 20bp (18–24 bp), and a GC content between 40% and 60%. The maximum allowed self-complementarity was restricted to minimize primer-dimer formation.