Table 2.

Evaluated primer sets. SS: second-strand synthesis; RT: reverse transcription; 5PCR2 and 3PCR2: 5′ and 3′ primers for the second amplification step (see Fig. 1 for further details).

Primer set	Positiona	Primer	Sequence	Amplicon sizeb (bp)
1	16,427–16,550	1_SS	AGCAAGATCCAATCTAGASTGATG	155
		1_5PCR2	CAAGATCCAATCTAGASTGATGA
		1_RT	CCAAGGGATTCCCAACCTYCCAAC
		1_3PCR2	AAGGGATTCCCAACCTYCCAACA
2	16,413–16,550	2_SS	GCAAGATCCAATCTAGAATGATSA	169
		2_5PCR2	AAGATCCAATCTAGAATGATSATT
		2_RT	CCAAGGGATTCCCAACCTYCCAAC
		2_3PCR2	AAGGGATTCCCAACCTYCCAACA
3	16,545–16,681	3_SS	AGCAAGATCCAATCTAGARTGTTG	168
		3_5PCR2	CAAGATCCAATCTAGARTGTTGG
		3_RT	AGGGATTCCCAACCTTCATCNACA
		3_3PCR2	GGATTCCCAACCTTCATCNACAAA
4	19,497–19,579	4_SS	AGCAAGATCCAATCTAGACARTGG	114
		4_5PCR2	CAAGATCCAATCTAGACARTGGGG
		4_RT	CCAAGGGATTCCCAACCTYNGCAT
		4_3PCR2	AAGGGATTCCCAACCTYNGCATC
5	1,949,719,588	5_SS	AGCAAGATCCAATCTAGACARTGG	123
		5_5PCR2	CAAGATCCAATCTAGACARTGGGG
		5_RT	CCAAGGGATTCCCAACCTCKWGTCA
		5_3PCR2	AAGGGATTCCCAACCTCKWGTCAT
6	19,959–20,032	6_SS	AGCAAGATCCAATCTAGATTTGAYA	105
		6_5PCR2	CAAGATCCAATCTAGATTTGAYAC
		6_RT	CCAAGGGATTCCCAACCTAANGCA
		6_3PCR2	AAGGGATTCCCAACCTAANGCATG
7	21,507–21,568	7_SS	AGCAAGATCCAATCTAGANNATGC	93
		7_5PCR2	CAAGATCCAATCTAGANNATGCC
		7_RT	CCAAGGGATTCCCAACCTCCATART
		7_3PCR2	AAGGGATTCCCAACCTCCATARTT
8	21,564–21,631	8_SS	AGCAAGATCCAATCTAGAAAYTAT	99
		8_5PCR2	CAAGATCCAATCTAGAAAYTATGG
		8_RT	CCAAGGGATTCCCAACCTTAYTGA
		8_3PCR2	AAGGGATTCCCAACCTTAYTGACA

^aPosition in alignment.

^bThe expected amplicon size for HCoV-NL63.