Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

Table 1 Primers and probes used in the multiplex qRT-PCR assay

Primers/probes	Sequences (5′-3′)^a	Genome positions^b	Final concentration (nM)
H7-F	GGRAAATGYCCRAGATATGTTAA	934–956	600
H7-R	AATTAGKCCTTCCCATCCATTT	1062–1083	600
H7-P-W	ROX-TTGGTGCYATAGCDGGTTTCATTGA-BHQ2	1037–1061	600
H7-P-M	FAM-AGRGAAAACGGRYTGCGA-MGB	1010–1027	800
N9-F	GAAGAATGCTCATGTTACGG	832–851	400
N9-R	TGACTAGTGTGTGTCATTGCTA	929–950	400
N9-P-W	Cy5-ATTGGCAGGGCTCAAATAGACCAGT-BHQ3	887–911	400
N9-P-M	VIC-GCACATGCAAGGACA-MGB	872–886	400

^aFAM, 6-carboxyfluorescein; Cy5, cyanine 5; ROX, 6-carboxy-X-rhodamine; MGB, minor groove binding; BHQ, Black Hole Quencher; Y = T or C, R = A or G, K = G or T, D = A or T or G
^bNucleotide position is based on the ORF of HA and NA genes of A/Anhui/1/2013(H7N9) (GISAID accession: EPI_ISL_138739) except the probe H7-P-M, which is based on the HA gene of A/Guangdong/Th005/2017(H7N9) (GISAID accession: EPI_ISL_250424)

ISSN: 1471-2334