Figure 2

Amplification, melting curve, and agarose gel electrophoresis of RT-PCR targeting a 220-bp product on ISAV segment 8 using total RNA from haemagglutination tests at different sampling points. (A) Real time RT-PCR amplification curves (water (Ct = 0) – no template negative control; 1% rbc rt (Ct = 0) – rainbow trout erythrocyte control; 1% rbc as(Ct = 0) – Atlantic salmon erythrocyte control; 0 hr rt (Ct = 28.6 ± 4.62) – NBISA01 haemagglutination reaction of 1% rainbow trout erythrocyte sampled at 0 hour; 0 hr as(Ct = 30.8 ± 0.70) – NBISA01 haemagglutination reaction of 1% Atlantic salmon erythrocyte sampled at 0 hour; 18 hr rt (sample not run in triplicate because of insufficient total RNA) – NBISA01 haemagglutination reaction of 1% rainbow trout erythrocyte sampled after 18-hour incubation; 18 hr as (Ct = 32.62 ± 1.10) – NBISA01 haemagglutination reaction of 1% Atlantic salmon erythrocyte sampled after 18-hour incubation; 36 hr rt (Ct = 30.27 ± 0.63) – NBISA01 haemagglutination reaction of 1% rainbow trout erythrocyte sampled after 36-hour incubation; 36 hr as (Ct = 27.59 ± 0.70) – NBISA01 haemagglutination reaction of 1% Atlantic salmon erythrocyte sampled after 36-hour incubation; NBISA01 (Ct = 28.46 ± 0.52) – virus positive control). (B) melting curve of the real time RT-PCR of the run in (A). (C) PCR products resolved on 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The order of the lanes in the gel picture is the same as in the amplification curves (A).