Based on recommendation for LAMP: the Tm ranged from 59 to
66 C; to assist with stability the ends of the primers were
designed to meet the requirement of free energy (DG) less
than -4 kcal/mol; GC content of primers was between 40
and 65 %; the distance ranged from 120 to 160 bases between
the inner primer set; and the distance of the inner
and outer primers ranged from 0 to 60 bases. Additionally,
at least one primer was designed to span intron/exon
junctions to prevent the amplifications from any contaminating
DNA. A series of primer sequences were obtained
and the optimal primer sets were selected from them
after trials.