TABLE 1.

Primer/probe sequences, thermocycler parameters, and purpose for each PCR assay

Purpose and oligonucleotide, probe, or primer	Sequencea	Thermocycling parameters
AfaDV quantification (qPCR)
Standard	TTCGTAATAGCACCTTCGTCACCAGCTAAATATAGATTTTCTCCAACTCTGATGAAACCAGCACGTCCACCAGACTGATGAATGCGCAGTAATTGTCTA	1 cycle at 50°C for 2 min, followed by 95°C for 2 min and 50 cycles of 95°C for 15 s and 58°C for 1 min (SsoAdvanced universal probes supermix used)
Internal probe	FAM-TGATGAAACCAGCACGTCCACCAGA-TAMRA
L-primer position 5664	CGTAATAGCACCTTCGTCACC
R-primer position 5738	GACAATTACTGCGCATTCATCA
AfaDV genome verification (PCR)
L-primer 1 position 243	TTTGAGGTCATATGGGCGGA	1 cycle at 94°C for 2 min; 30 cycles of 45 s at 94°C, 30 s at 56°C, and 45 s at 72°C; and a final extension step for 2 min at 72°C (Taq DNA polymerase used)
R-primer 1 position 833	CTTGTCACAACTCCTTTTCGC
L-primer 2 position 554	ACGCCACTCAGTATGCAGTA
R-primer 2 position 1061	TCCCAAGCTTTGCCAGAGTA
L-primer 3 position 831	TGCGAAAAGGAGTTGTGACA
R-primer 3 position 1361	TGCAAACGCTATCTTCTTCTCC
L-primer 4 position 1266	TGCCGGATCTGACCATTGAT
R-primer 4 position 1702	TTCTCGACATACCTGGAGCA
L-primer 5 Position 1520	AAGCAGCAAAGACATGGAGC
R-primer 5 position 2075	GATCCGGTTCGTCATCATCG
L-primer 6 position 1979	GGAGAGCGGACTTGATGGAT
R-primer 6 position 2564	AGAAATTCTTACCCGCTGAAGG
L-primer 7 position 2378	GTGCAGGGTACGGTAATTTTG
R-primer 7 position 2919	ACAGCAAGCGGATTAGGTTTC
L-primer 8 position 2655	CCATTTCAAGACGCTGAGGG
R-primer 8 position 3144	AATGTTGCTCCACCAGTTGC
L-primer 9 position 3066	CTTGGGCGAGTCATACGAGA
R-primer 9 position 3599	AGTCTGTTGGAAACGCTCAG
L-primer 10 position 3440	AGCAGAGTCACCACGAACAT
R-primer 10 position 3895	CGGTACTGATCAATCTTCTGCT
L-primer 11 position 3707	TGATCCCAAGTAGTATCGTTCG
R-primer 11 position 3999	ATGAGAGGAGGAGTCGATAGG
L-primer 12 position 3895	AGCAGAAGATTGATCAGTACCG
R-primer 12 position 4397	ATTCGCAAAGTGATGGAGGC
L-primer 13 position 4215	TGGGATTTTAGCGAGAGGAGT
R-primer 13 position 4770	AGATCACGTCCTAGTAGTGCT
L-primer 14 position 4582	CACCTTCAGCTTGGCGTATA
R-primer 14 position 5031	TCTTCCTCAGGTATGTCGCA
L-primer 15 position 4914	TGTTGGCCCTTTTGAGTAGG
R-primer 15 position 5461	TGTTGCTGCTGGTACTTCTG
L-primer 16 position 5257	TCGTCATCAACATCAACAGGC
R-primer 16 position 5866	TTTGAGGTCATATGGGCGGA
AfaDV oocyte and pyloric caecum detection, VP mRNA (PCR/RT-PCR)
L-primer 9 position 3066	CTTGGGCGAGTCATACGAGA	1 cycle at 98°C for 30 s; 35 cycles of 10 s at 98°C, 20 s at 66°C, and 20 s at 72°C; and a final extension step for 2 min at 72°C (Q5 polymerase used)
R-primer 9 position 3599	AGTCTGTTGGAAACGCTCAG
AfaDV VP cloning (PCR)
L-primer position 252 (restriction enzyme EcoRI)	CGCgaattcATAGAAAAGGCTGTG	1 cycle at 98°C for 30 s; 30 cycles of 10 s at 98°C, 30 s at 66°C, and 1 min 30 s at 72°C; and a final extension step for 2 min at 72°C (Q5 polymerase used)
R-primer position 3187 (restriction enzyme HindIII)	CGCaagcttCCTAATCCGCT
SSaDV VP cloning (PCR)
L-primer position 2857 (restriction enzyme HindIII)	GGGGaagcttAGAAACCTAATCC	1 cycle at 98°C for 30 s; 30 cycles of 10 s at 98°C, 30 s at 67°C, and 1 min 30 s at 72°C; and a final extension step for 2 min at 72°C (Q5 polymerase used)
R-primer position 5731 (restriction enzyme EcoRI)	CTGAgaattcCATTATGTCGGGTG
AfaDV NS1, NS2, and NS3 cloning (PCR)
L-primer 1 position 243	TTTGAGGTCATATGGGCGGA	1 cycle at 98°C for 30 s; 30 cycles of 10 s at 98°C, 30 s at 67°C, and 1 min 30 s at 72°C; and a final extension step for 2 min at 72°C (Q5 polymerase used)
R-primer 8 position 3144	AATGTTGCTCCACCAGTTGC
SSaDV (PCR)
SSaDV_NS3_1_F	CAATACGCCGATTAGCTTACAG	1 cycle at 98°C for 30 s; 35 cycles of 10 s at 98°C, 30 s at 64°C, and 40 s at 72°C; and a final extension step for 2 min at 72°C (Q5 polymerase used)
SSaDV_NS2_1_R_2	TCCTCGCTCACTACTAATGTTG

^aLowercase type in sequences indicates the restriction enzyme. FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.