Colibri spectrometer

Several duplex PCR mixtures and thermocycler conditions were trialled in this study (data not shown) in order to determine optimal PCR conditions, which were finally set up as follows. The PCR reaction mixture was composed of 12.5 µl Master Mix (Go-Taq ® Green, Promega USA), 0.8 µM of each primer F11 and R5, 0.2 µM of each primer FnoF1 and FnoR1, 4 µl of DNA template (150-200 ng) and nuclease-free water in a final volume of 25 µl. Thermocycler conditions were performed as follows: denaturation at 94°C for 3 min; 35 cycles of amplification at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min; final extension at 72°C for 5 min. PCR products were then electrophoresed with 1% agarose gel and visualized under UV light The specificity of the duplex PCR assay was tested with DNA extracted from 14 bacterial isolates recovered from diseased fish as mentioned above (Table 1). Sensitivity testing of the PCR assay was performed with 10-fold serial dilutions (200 ng to 0.2 fg) of genomic DNA from the 2 bacterial isolates VMCU-FNO131 and AL1104 (Table 1). DNA concentration of each bacterial isolate was quantified using Colibri Spectrometer (Titertek Berthold, Germany) and adjusted to the desired concentrations mentioned above.