Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction

Table 2 Primer and probe oligonucleotides for qRT-PCR assays used in this study

Assay/Oligo	Sequence (5′ to 3′)	Final conc. (μM)
AI Type A
IVA D161M	AGATGAGYCTTCTAACCGAGGTCG	0.9
IVA D162M1	TGCAAAAACATCYTCAAGTCTCTG	0.225
IVA D162M2	TGCAAACACATCYTCAAGTCTCTG	0.225
IVA D162M3	TGCAAAGACATCYTCAAGTCTCTG	0.225
IVA D162M4	TGCAAATACATCYTCAAGTCTCTG	0.225
IVA MA	(FAM)-TCAGGCCCCCTCAAAGCCGA-(TAMRA)	0.25
H5 duplex
IVA D148H5	AAACAGAGAGGAAATAAGTGGAGTAAAATT	0.675
IVA D149H5	AAAGATAGACCAGCTACCATGATTGC	0.675
IVA H5A	(FAM)-TCAACAGTGGCGAGTTCCCTAGCA-(TAMRA)	0.3
IVA D204f	ATGGCTCCTCGGRAACCC	0.675
IVA D205r	TTYTCCACTATGTAAGACCATTCCG	0.675
IVA D215P	(FAM)-ATGTGTGACGAATTCMT-(MGB-NFQ)	0.3
H5 2.3.4.4-BHQ1
D-718 FWD	CAAGAAAGGGGACTCAACAATTATG	0.3
D-719 REV	TGAGAGGGTGTATATTGTGGAATGG	0.9
D-720 PRB	(FAM)-TTGACATTTGGTGTTGCAGTGGCCATA-(BHQ1)	0.25
D-720 PRB	[TTGACATTTGGTGTTGCAGTGCCCATA*]	0.25
H5 2.3.4.4-MGB
D-718 FWD	CAAGAAAGGGGACTCAACAATTATG	0.3
D-719 REV	TGAGAGGGTGTATATTGTGGAATGG	0.9
D-724 PRB (MGB)	(FAM)-ATGGCCACTGCAAC-(MGB-NFQ)	0.25
D-724 PRB (MGB)	[ATGGGCACTGCAAC*]	0.25
AI N6
D-721 FWD	CCCCACCAATGGGAACTG	0.9
D-722 REV	TCTAGGAATGCAAACCCTTTTACC	0.3
D-723 PRB	(FAM)-CCAATAACAGGAGGGAGCCCAGACCC-(BHQ1)	0.25

*H5N6 2.3.4.4 virus with a single nucleotide mismatch in the TaqMan probe sequences has also been recently detected. Accordingly, to ensure greatest potential future utility additional probe sequences containing the single nucleotide substitution are given in bold.

ISSN: 1743-422X