Sensitivity of the Dirofilaria Immitis nested PCR (A) and Wolbachia nested PCR (B) assays. Precise serial dilutions of the linearized pCR-4 TOPO vector containing the D. immitis COI and Wolbachia FtsZ sequences were used as a PCR template. The amounts of target are indicated above each lane. (A) Lanes 2 to 9, first PCR round. Upper bands of the expected 634-bp size represent the Fil_COIclon amplicons, whereas the lower 280-bp bands represent the mimic internal standard. Lanes 11 to 15, second PCR round. Upper bands of the expected 406-bp size represent the COIfel amplicons, whereas the lower 237-bp bands represent the mimic internal standard. (B) Lanes 2 to 9, first PCR round. Upper bands of the expected 267-bp size represent the Wol1 amplicons, whereas the lower bands of 220 bp represent the mimic internal standard. Lanes 11 to 15, second PCR round. Lower bands of the expected 147-bp size represent the Wol7 amplicons, whereas the upper 309-bp bands represent the mimic internal standard. MWM, molecular weight marker, 100-bp ladder. The 500-bp band is indicated by an arrowhead.