Division II.1.2 RT-PCR for ISAV detection

Several RT-PCR methods may be used for ISAV genome amplification. A two-step RT-PCR may be performed whereby the RT and the PCR reactions steps are run in two separate tubes. However, a one-step reaction, where the two reactions are run in one tube, may also be performed. The one-step method shall be used where possible, as the one tube assay minimises the risk of cross-contamination as no transfer of content have to be made and it is regarded to be as sensitive as the two-step method.

The primers and assay described in this point, namely the ILA1 or ILA2 primer pair that target segment 8 and which have been found suitable for detection of ISAV in outbreaks and in carrier fish, shall be used. The ILA2 reverse primer does not match isolates from North America and an alternative primer set shall be used in those cases.

• Forward primer (ILA1): 5′-GGCTATCTACCATGAACGAATC-3′;

• Reverse primer (ILA2): 5′-GCCAAGTGTAAGTAGCACTCC-3′.

Cycling conditions: one cycle of 50 °C for 30 minutes, 1 cycle of 94 °C for 15 minutes, 40 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 60 seconds; one cycle of 72 °C for 5 min. Product size 155 bp.

The results of the PCR may vary depending on the conditions under which it is performed, namely the thermal protocols might need optimisation, depending on the thermal cycler in use. Furthermore, false-positive results may occur because of false primer annealing or laboratory contamination. Negative template controls and positive controls shall be included on each plate run. However, other RT-PCR versions of proven similar efficacy may be used instead.