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Table 1 Oligonucleotide primers and probes designed for this study

From: Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper) and escape mutant (clade 2.2.1 variant) lineages in Egypt

No. Primers/probes Sequence 5'- 3' Conc.1 (nM) Position2 Amplificate size (bp)
1 P1FW_2.2.1p GAR TCA ATA GGA AYT TAC CAA ATA CTG 400 1615-1641 85
2 P1FW_2.2.1v GAA TCA ATA GGA ACT TAC CAA ATA CTA TC 800 1615-1643  
3 P1RV_2.2.1 AGA CCA GCC ACC ATT GAT TGC 400 1699-1679  
4 PRO1.1_2.2.1v FAM-ACA GT G GCA AGT TCC CT-BHQ-1 64 1654-1670  
5 PRO1.2_2.2.1p HEX-ACA GTG GCG AGC TCC CTA GC-BHQ-1 64 1652-1675  
6 P2FW_2.2.1p GGA TTC ACC ATC CR A ATG ATGC 1600 620-641 106
7 P2FW_2.2.1v GGG ATT CAC CAT CCA AAT GAT GA 1600 619-641  
8 P2RV_2.2.1p CCG TTT ACC TTA GAT CTA GTA GCT ATT 1600 752-726  
9 P2RV_2.2.1v CCG TTT ACC TTA GAT CTA GTR GCT ATC 1600 752-726  
10 PRO2_2.2.1 ROX -TAC CTA TAT TTC CGT TGG GAC ATC AAC ACT AAA-BHQ-2 64 675-707  
  1. 1 Nanomolar concentration of a 25 μl reaction.
  2. 2Position relative to the initiating codon of A/chicken/Egypt/06541-NLQP/2006 (H5N1), GenBank accession no. EU372946.1
  3. Bold face nucleotides indicate mismatch positions between clade 2.2.1p proper and lineage 2.2.1v variant viruses. Italics indicate use of locked nucleic acid nucleotides.
  4. The specificity of primers and probes for either or both of the 2.2.1 lineages is indicated with their designation.