Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes

Table 1 Primers and probes of the qq-PCR assay

Targets	Gene	NCBI ID of reference sequence	Genome position	Primer (probe)	Sequence (5′–3′)	Final concentration (μM)
SARS-CoV-2	S	NC_045512.2	25,197–25,324	Primer-F1	GGCCATGGTACATTTGGCT	1.2
				Primer-R1	GCAGCAGGATCCACAAGAA	1.2
				Probe 1	FAM-TTGCTGTAGTTGTCTCAAGGGCTG-BHQ-1	0.6
SARS-CoV-2 and SARS	N	NC_045512.2	28,677- 28,778	Primer-F2	CTGAGGGAGCCTTGAATACA	1.2
				Primer-R2	TTGGCAATGTTGTTCCTTGA	1.2
				Probe 2	ROX-AACAATGCTGCMAYCGTGCTAC-BHQ-2	0.6
HCoV-229E	ORF1ab	NC_002645.1	7464–7568	Primer-F3	ACTTTGTCAGTTCGTATGCTAAAC	1.2
				Primer-R3	TTGTCAGAACATTGGCATTAACA	1.2
				Probe 3	Cy5-ATTATCAGCTTATGACTTGGCGTGT-BHQ-2	0.6
HCoV-NL63	ORF1ab	NC_005831.2	23,263–23,376	Primer-F4	GTTCTCTTATAGGTGGCATGGT	1.2
				Primer-R4	GAAGCACATCAGTTTGTAAAGCA	1.2
				Probe 4	Cy5-CCTTTTTCTTTGGCACTGCAAGCAC-BHQ-2	0.6
HCoV-HKU1	ORF1ab	NC_006577.2	6671–6762	Primer-F5	AGTTTATCTTTAGTTGATGTTTGGGA	1.2
				Primer-R5	GATTTAACTAGGCGTGACAATTCAT	1.2
				Probe 5	Cy5-TATTTGACAGGTTGTGATTATGTTGTTTGGG-BHQ-2	0.6
HCoV-OC43	ORF1ab	NC_006213.1	12,518–12,631	Primer-F6	GTGGCCACTAGTTATTATTGCAAA	1.2
				Primer-R6	TCTGGACCACTATTAACAACCTG	1.2
				Probe 6	Cy5-TTGCAAAATAATGAATTAATGCCTGCTAAGTTGA-BHQ-2	0.6
human RNA	B2M	NM_004048.4	92–238	Primer-F7	TCCAGCGTACTCCAAAGATT	1.2
				Primer-R7	CCACTTTTTCAATTCTCTCTCCATT	1.2
				Probe 7	VIC-TCTGCTGGATGACGTGAGTAAACCTG-BHQ-2	0.6

SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SARS-CoV, severe acute respiratory syndrome coronavirus; HCoV, human coronavirus

ISSN: 1743-422X