Limit of detection of Opisthorchis viverrini DNA by pPCR (A) and nPCR (B) on agarose gels (1.5%) stained with ethidium bromide. Lane M, DNA size marker (1-kb DNA ladder; Merck); lane N: negative controls containing no DNA. (A) Primary primers tested in 10-fold serial dilutions with O. viverrini DNA at 7.5 × 104 pg (lane 1), 7.5 × 103 pg (lane 2), 750 pg (lane 3), 75 pg (lane 4), 7.5 pg (lane 5), and 0.75 pg (lane 6). The size of the amplicon was 508 bp. (B) Nested primers amplifying the pPCR product of serial dilutions of O. viverrini DNA at 75 pg (lane 1), 7.5 pg (lane 2), 0.75 pg (lanes 3 to 5), 7.5 × 10−2 pg (lanes 6 to 8), and 3.75 × 10−2 pg (lanes 9 to 11). The size of the amplicon was 226 bp.