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

Table 1 Oligonucleotide primers and probes designed for this study

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 Nanomolar concentration of a 25 μl reaction.
²Position relative to the initiating codon of A/chicken/Egypt/06541-NLQP/2006 (H5N1), GenBank accession no. EU372946.1
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.
The specificity of primers and probes for either or both of the 2.2.1 lineages is indicated with their designation.

ISSN: 1743-422X