TABLE 3.

Primers and standard sequences used for two-step RT-qPCR to detect PAA-induced genome damage of RV and TV

Target gene	Step no.a	Primer name	Sequence (5′–3′)	Position in the gene (nt)	Amplicon size (bp)	PCR conditions
RV strain OSU VP7b	1	OSU-VP7-R	GTTAGAACTGTATGATGTGACC	1041–1062	1,052	42°C for 60 min and 80°C for 5 min
	2	OSU-VP7-qPCR-F1	GAGAGAATTTCCGACTGG	11–28	131	95°C for 10 min; then 40 cycles of 95°C for 15 s and 60°C for 1 min
		OSU-VP7-qPCR-R1	GTCCATTGTTCTAGTAACTGA	121–141
TV NSPc	1	TV-NSP-R	TGAGGTCTTCTTCAACGCCC	4078–4098	1,324	42°C for 60 min and 80°C for 5 min
	2	TV-NSP-qPCR-F	GGCAGCTGGGAAGAAATCTG	2774–2794	111	95°C for 10 min; then 40 cycles of 95°C for 15 s and 60°C for 1 min
		TV-NSP-qPCR-R	CCTGCTGTGTGAATGCCTAC	2865-2885

^aStep 1 consists of reverse transcription of the target gene, and step 2 consists of qPCR of the target gene.

^bqPCR standard sequence (5′–3′) acquired from GenBank accession number KJ450849.1: GGCTTTAAAAGAGAGAATTTCCGACTGGCTATCGGATAGCCTTTTTAATGTATGGTATTGAATATACCACAGTTCTAACTTTTTTGATATCGCTTGTATTTGTCAATTATATACTGAAATCAGTTACTAGAACAATGGACTTTATCATTTATAGATTCTTATTGGTTATAGTCGTACTTGCACCGCTCA. The forward (F) primer attaches to the sequence indicated in boldface, and the reverse primers (R) attach to the complementary strand indicated by underlining.

^cqPCR standard sequence (5′–3′) acquired from GenBank accession number EU391643.1: CCGTGGTTGTGCGCAGTATTGGAAACACAAACATTGCTGGGAAATTCCTCAACGTCTTCACAGGTACAGTTGTGGCAGCTGGGAAGAAATCTGACGGCCTCGGGTCTGAACCAGGAGACTGTGGCTCACCATATCTTAAATTTGTTAATGGAAAACCAACTCTTGTAGGCATTCACACAGCAGGCAGCTACACTACCAACCAGGTTGCAGGCTTAGTGATACCTTCTAGATTCAACCTTG. The forward (F) primer attaches to the sequence indicated in boldface, and the reverse primers (R) attach to the complementary strand indicated by underlining.