Division II.2.2 Conventional PCR for confirmation of KHV detection

United Kingdom

For confirmation of the presence of infection with KHV, the generic nested PCR described in the following Table 2.1 shall be used followed by sequencing of the amplified product.

Table 2.1

Primers and conditions for the nested PCR assay targeting all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3)

Primer name	Sequence	Cycling conditions	Product size
CyHVpol-forward	5′-CCAGCAACATGTGCGACGG-3′	First round PCR 1 cycle: 95 °C 2 minutes 40 cycles: 95 °C for 30 seconds 55 °C for 30 seconds 72 °C for 45 seconds 1 cycle: 72 °C for 10 minutes	362 bp
CyHVpol-reverse	5′-CCGTARTGAGAGTTGGCGCA-3′		362 bp
CyHVpol-internal forward	5′-CGACGGVGGYATCAGCCC-3′	Second round PCR 1 cycle: 95 °C 2 minutes, 40 cycles: 95 °C for 30 seconds 55 °C for 30 seconds 72 °C for 45 seconds 1 cycle: 72 °C for 10 minutes	339 bp
CyHVpol-internal reverse	5′-GAGTTGGCGCAYACYTTCATC-3′		339 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. PCR versions of proven similar efficacy may be used instead.

Sequencing may be performed by the laboratory or at external specialised sequencing companies. Sequencing results shall be analysed by aligning the sequences to the known reference sequences of KHV (Gen Bank accession numbers AP008984, DQ657948 and DQ177346). The obtained clean consensus sequence must match at least to 98 % with those reference sequences.