Division I.2.2 Conventional PCR for KHV detection

The assay described in this point targeting the Thymine kinase (TK) gene of KHV shall be used. However, other PCR assays with demonstrated similar sensitivities and specificities to the described assay may be used instead.

• Forward primer (KHV-TKf): 5′-GGGTTACCTGTAC GAG-3′;

• Reverse primer (KHV-TKr): 5′-CACCCAGTAGATTA TGC-3′.

Cycling conditions: one cycle of 95 °C for 5 minutes followed by 35 cycles of 95 °C for 30 seconds, 52 °C for 30 seconds, 72 °C for one minute and one cycle of 72 °C for 10 minutes. Product size should be 409 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 contamination. Negative template controls and positive controls shall be included on each plate run. However, other PCR versions of proven similar efficacy may be used instead.

The first detection in an area shall be confirmed by sequencing or sent to a to a national reference laboratory F1... for immediate identification.