. 2016 Jan 11;4:e1560. doi: 10.7717/peerj.1560

Table 1. Primers and probes designed for multiplex qRT-PCR rotavirus genotyping assay.

	Target real-time assays	Nucleotide sequence (5′-3′)^a	Fluorophore-quencher (5′-3′)	Conc./qRT-PCR reaction (nM)	Nucleotide position (bp)	Tm (°C)	Amplicon length (bp)
Well 1	RotaTeq® VP6	FP^b-GCGGCGTTATTTCCAAATGCACAG		200	Gautam et al., 2013
		RP^c-CGTCGGCAAGCACTGATTCACAAA		200
		Probe^e-ATCACGCAA″C″AGTAGGACT″C″ACGCTT	HEX-BHQ1	100
	Xeno-IPC	FP^b-Proprietary		400	Proprietary
		RP^c-Proprietary		400
		Probe^e-Proprietary	TR-BHQ2	100
	MS2-IPC	FP^b-TGGCACTACCCCTCTCCGTATTCACG		400	289–314	63	99
		RP^c-GTACGGGCGACCCCACGATGAC		400	387–366	64
		Probe^e-CACATCGATAGATCAAGGTGCCTACAAGC	TR-BHQ2	200	330–358	60
	Rotarix® NSP2	FP^b-GAACTTCCTTGAATATAAGATCACACTGA		400	Gautam et al., 2013
		RP^c-TTGAAGACGTAAATGCATACCAATTC		400
		Probe^e-TCCAATAGATTGAAGT{C}AGTAA″C″GTTTCCA	Cy5-BHQ3	200
	G12	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		200	37–59	57	309
		RP^c-CGTCCARTCRGGRTCAGTTATTTCAGTC		200	345–318	60
		Probe^d-ARTTTTGAG{C}TYYAATAAATGGCAGYAYG	FAM-BHQ1	100	205–177	65
Well 2	G9	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		600	37–59	57	318
		RP^c-CAGAGTATYYTT″C″CATTCHGTATCTCC		600	354–328	58
		Probe^e-CCACARTT{C}TAA{C}CTTTYTGATATCA	HEX-BHQ1	200	68–93	68
	NSP3	FP^b-ACCATCTWCACRTRACCCTC		400	Mijatovic et al., 2013
		RP^c-GGTCACATAACGCCCCTATA		400
		Probe^e^,^f-ATGAGCACAATAGT″T″AAAAGCTAACACTGTCAA	TR-BHQ2	100
	G4	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		600	37–59	57	319
		RP^c-ATAGWGTAT{C}TTTCCATTCAKTGTC		600	355–331	59
		Probe^e-TGTAGTATTRT{C}AKTATTAK{C}GAATGCRC	FAM-BHQ1	200	171–199	70
Well 3	G1	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		400	37–59	57	305
		RP^c-CAKTCACCATCATTRATTTGAGTACTTGCT		400	341–322	57
		Probe^d-TCCATTGAT{C}CTGTTATTGGTAAGTTAAG	HEX-BHQ1	100	239–210	63
	P4	P-consensus-FP^b-GG″C″TATAAAATGG″C″TTCGCT		400	1–20	59	459
		RP^c-ATYACYMTGACTACTAC″C″TTTAAA″C″ATTTCG		400	459–429	60
		Probe^d^,^f-ATGTGGT″T″CRACWGCGATAACTG″C″TGTC ″C″AAAA	TR-BHQ2	100	342–310	69
	G3	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		400	37–59	57	459
		RP^c-TTGCAGTGTAG″C″GTCRTAYTTC		400	495–474	58
		G3-P^e-TGTATTAYC{C}AACTGAAG″C″WGCAACAG	Cy5-BHQ3	100	296–322	69
Well 4	G2	G-consensus-FP^b-TAG{C}TCYTTTTRATGTATGGTAT		200	37–59	57	352
		RP^cCARTYGGCCAT{C}CTTTAGTTA		200	388–378	60
		Probe^e-CCAATAACGGGRT{C}ACTAGACGCTGT	TR-BHQ2	100	220–246	69
	P8	P-consensus-FP^b-GG″C″TATAAAATGG″C″TTCGCT		400	1–20	59	424
		RP^c-ACTTCCAYTTAT{C}TGAATCRTTWCT		400	424–400	60
		Probe^e-A{C}TGYAGTMGTTG″C″TRTTGAACCRCA	Cy5-BHQ3	200	316–341	70
		P6	P-consensus-FP^b-GG″C″TATAAAATGG″C″TTCGCT		400	1–20	59	297
	RP^c-AAGCAAYCCAAAYAT{C}AGTTTTATTG			400	297–322	68
	Probe^eTGAATC{C}AACTAATCAA{C}AAGTTG		FAM-BHQ1	200	260–283	68

Notes:

^a {C}, AP-dC (G-clamp); “C,” C-5 propynyl-dC (pDC); “Y”-C or T; “R”-A or G; “M”-C or A; “K”-T or G; “W”-T or A.

^b Forward primer.

^c Reverse primer.

^d Probe in reverse compliment orientation.

^e Probe in forward orientation.

^f Probe with internal quencher at “T” nucleotide. IPC-Internal Positive Control.

HEX-5′-Hexachloro-Fluorescein Phosphoramidite- (Glen Research, Catalog Number- 10-5902-95).

CalRed 610 or Texas Red (TR) - (BioSearch Technologies, Catalog Number-BNS-5082-50).

Quasar 670 or Cy5- (BioSearch Technologies, Catalog Number-FC-1065-100).

FAM-5′ carboxyfluorescein- (Glen Research, Catalog number- 10-5901-95).

BHQ1- Black Hole Quencher-1-(BioSearch Technologies, Catalog Number-3-BHQ1-1).

BHQ2-Black Hole Quencher-2-(BioSearch Technologies, Catalog Number-CG5-5042G-2).

BHQ3-Black Hole Quencher-3-(BioSearch Technologies, Catalog Number-CG5-5043G-2).