Supporting File1. Primer and probedesigns for virus-specific PCR assays.

RNA viruses weredetectedusing specific primersand probes. PCR amplification wasperformed in a 25μLreaction,including 5 μL ofRNA templateand 20 μL ofPCR master mix, containing 400 nM primers and200 nM TaqMan probes. Real-time PCR was performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA).Conventional PCR was performed on a Biometra T Gradient PCR machine (Biometra, Göttingen, Germany) underthe following conditions: 50°C for 30 min, 95°C for 10 min, 45 cycles of 95°C for 20 s, and 60°C for 1min. Amplified products were sequenced using the BigDye Terminator Cycle-Sequencing Kit(Thermo Fisher Scientific, Waltham, MA, USA).For human rhinovirus, subtype-specific primers targeting the VP4/VP2 region were designed, followed by queriesagainstthe NCBI nucleotide database for genotyping. For enterovirus (EV), viral detectionand sequencing were performed using EV CODEHOP primers, followed by NCBI queries for genotyping. All primer sequences are listed below.

Virus; target gene; amplicon size in bp [reference] / Primer forward (F), reverse (R), or probe (P) / Sequences (5′ to 3′)
EV; VP3; 993 [1] / F / GCIATGYTIGGIACICAYRT
R / CICCIGGIGGIAYRWACAT
EV; VP1; 376 [1] / F / CCAGCACTGACAGCAGYNGARAYNGG
R / TACTGGACCACCTGGNGGNAYRWACAT
HCoV-229E; NP; 76 [2] / F / CAGTCAAATGGGCTGATGCA
R / AAAGGGCTATAAAGAGAATAAGGTATTCT
P / FAM-CCCTGACGACCACGTTGTGGTTCA
HCoV-NL63; NP; 109 [3] / F / GACCAAAGCACTGAATAACATTTTCC
R / ACCTAATAAGCCTCTTTCTCAACCC
P / Cy5AACACGCT"T"CCAACGAGGTTTCTTCAACTG-phosphate
HCoV-OC43; NP; 75 [2] / F / CGATGAGGCTATTCCGACTAGGT
R / CCTTCCTGAGCCTTCAATATAGTAACC
P / HEX-TCCGCCTGGCACGGTACTCCCT
hMPV-2; NP; 162 [4] / F / CATAYAARCATGCTATATTAAAAGAGTCTC
R / CCTATYTCWGCAGCATATTTGTARTCAG
P / FAM-AATGATGARGGTGTCACTG-MGBNFQ
hMPV-2; HN; 115 [5] / F / CCATTTACCTAAGTGATGGAA
R / CGTGGCATAATCTTCTTTTT
P / Texas Red-AATCGCAAAAGCTGTTCAGTCAC
HPIV-3; HN; 153 [5] / F / GGAGCATTGTGTCATCTGTC
R / TAGTGTGTAATGCAGCTCGT
P / FAM-ACCCAGTCATAACTTACTCAACAGCAAC
HPIV-4; HN; 199 [5] / F / CCTGGAGTCCCATCAAAAGT
R / GCATCTATACGAACACCTGCT
HPeV; 5’UTR; 143 [68] / F / CTGGGGCCAAAAGCCA
R / GGTACCTTCTGGGCATCCTTC
P / AAACACTAGTTGTAWGGCCC
HPeV; VP1; 759 [9] / F / CCAAAATTCRTGGGGTTC
R / AAACCYCTRTCTAAATAWGC
HRV; VP4/VP2; 642 or 539 [10] / F / CGGCCCCTGAATGYGGCTAA
F / CTACTTTGGGTGTCCGTGTTTC
R / ATCHGGHARYTTCCAMCACCA
IAV; MP; 104 [11] / F / AAGACCAATCCTGTCACCTCTGA
R / CAAAGCGTCTACGCTGCAGTCC
P / FAM-TTTGTGTTCACGCTCACCGT-TAMRA
RSV; L; 154 [5] / F / TTTCCACAATATYTAAGTGTYAA
R / TCATCWCCATACTTTTCTGTTA
P / HEX-CCATGTGAATTCCCTGCATCAAT
Rota-A; NSP3; 83 [12] / F / ACCATCTWCACRTRACCCTC
R / CACATAACGCCCCTATAGCC

EV=enterovirus; HCoV=human coronavirus; HMPV=human metapneumovirus; HPIV=human parainfluenza virus;
HPeV=human parechovirus; HRV=human rhinovirus; IAV=influenza A virus; RSV=respiratory syncytial virus; Rota=rotavirus

References

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