Division II.1.4 Sequencing of amplified PCR products

• Forward primer (ILAs6-3F): 5′-ATGAGGGAGGTAGCATTGCA -3′;

• Reverse primer (ILAs6-2R): 5′-CATGCTTTCCAACCTGCTAGGA -3′.

Negative template controls and positive controls shall be included on each plate run. Cycling conditions (One-step RT-PCR): one cycle of 50 °C for 30 minutes, one 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 minutes; it shall be adjusted if necessary. Other RT-PCR or RT-qPCR versions of proven similar efficacy may be used instead.

Alternatively, the following method for sequencing HPR in segment 6 may be used:

• Forward primer: 5′-GAC-CAG-ACA-AGC-TTA-GGT-AAC-ACA-GA-3′;

• Reverse primer: 5′-GAT-GGT-GGA-ATT-CTA-CCT-CTA-GAC-TTG-TA-3′;

• Product size: 304 nt if HPR0.

RT-PCR assays with similar sensitivities and specificities to the assays described in this point may also be used.

The purity of the amplified RT-PCR product shall be checked by gel electrophoresis before sequencing. If only one pure fragment appears, it shall be purified directly from the PCR reaction. If multiple amplified fragments are present, the fragment of interest shall be purified by gel electrophoresis. The purification of PCR fragments from solutions or agarose gels shall be made using PCR fragment affinity spin columns, according to the manufacturer's instructions.

Sequencing shall be performed using amplification primers at external specialised sequencing companies. The results shall be analysed with the search tool BLAST and the sequences shall be compared with other known sequences in the US National Centre for Biotechnical Information (NCBI) nucleotide database.

Sequencing must eliminate any doubt on the specificity of an amplified RT-PCR product.