Table 2. Oligonucleotides used for detection and quantification of cosaviruses*.

Oligonucleotide identity	Sequence, 5′ → 3′	Genomic target region, RT-PCR type	Use	Reference
DKV-N5U-F1	CGTGCTTTACACGGTTTTTGA (+)	5’-UTR, nested RT-PCR 1st round	Cosavirus detection†	(3)
DKV-N5U-R2	GGTACCTTCAGGACATCTTTGG (–)
DKV-N5U-F2	ACGGTTTTTGAACCCCACAC (+)	5’-UTR, nested RT-PCR 2nd round
DKV-N5U-R3	GTCCTTTCGGACAGGGCTTT (–)
HCosV-rtF735-1	TTGTAGYGATGCTGTRTGTGTGTG (+)	5’-UTR, real time RT-PCR	Brazilian cosavirus quantification†	This study
HCosV-rtP783	FAM-AGCCTCACAGGCCRRAAGCCCTGTC-DDQ1 (+, Probe)
HCosV-rtR827-1	CCAYTGTGTGGGTCCTTTCG (–)

*RT-PCR, reverse transcription PCR; UTR, untranslated region; FAM, fluorescein; R, G/A; DDQ1, deep dark quencher 1; Y, C/T.
†RT-PCR reactions were carried out using the QIAGEN One-step RT-PCR kit as described by the manufacturer (QIAGEN, São Paulo, Brazil), 300 nmol/L of each primer, 200 nmol/L of the probe (real time RT-PCR assay), 1 μg bovine serum albumin, and 5 μL RNA extract. Second-round reactions used the Platinum Taq DNA Polymerase Kit as described by the manufacturer (Invitrogen, São Paulo, Brazil) with 2.5 mol/L MgCl and 1 µL of first-round PCR product. Real time RT-PCR amplification involved 55°C for 15 min, 95°C for 15 min, and 45 cycles of 95°C for 15 s and 58°C for 30 s (fluorescence measured).Nested RT-PCR involved 30 min at 50°C; 15 min at 95°C; 10 cycles of 20 s at 94°C, 30 s starting at 60°C with a decrease of 1°C per cycle, and 50 s at 72°C; and 40 cycles of 20 s at 95°C, 30 s at 54°C, and 50 s at 72°C; and a final elongation step of 5 min at 72°C. Second-round reactions used 3 min at 94°C and thermal cycling as for the first round.