Table 5.

Oligonucleotide sequence of primers and probes used in Pan-Flavi qRT-PCR assay

Primer & Probe	Sequence	Concentration	Orientationa	Positionb	Tm (°C)
Flavi all S	TACAACATgATggggAARAgAgARAA	10 pmol	S	8993–9019	54.7
DEN4 F	TACAACATgATgggRAAACgTgAGAA	10 pmol	S	8996–9019	60.18
Flavi all AS 1	gTCCCANCCDgCKgTRTC	10 pmol	AS	9236–9253	52.5
	gTCCCATCCAgCKgTRTCATC	5 pmol	AS	9236–9256	57.1
Flavi all AS 2	gTgTCCCAgCCNgCKgTgTCATCWgC	10 pmol	AS	9232–9260	69.6
Flavi all probe 1	FAM-AARggHAgYMgNgCCA + TH + T + g + g + T-BBQ	5 pmol	S	9044–9065	69–83
Flavi all probe 2	FAM-Tg + gTWYATgTggYTNg + gRgC--BBQ	5 pmol	S	9062–9082	62–75
Flavi all probe 3 mix	FAM-Tg + gTWYATgT + ggYTNg + gRgC—BBQc	5 pmol	S	9062–9082	66-79
	FAM-CCgTgCCATATggTATATgTggCTgggAgC-BBQd	0.5 pmol	S	9052–9081	74.7
	FAM-TTTCTggAATTTgAAgCCCTgggTTT-BBQe	0.5 pmol	S	9086–9012	68
Flavi all S2	TACAACATgATgggMAAACgYgARAA	10 pmol	S	8996–9019	58.2
Flavi all AS4	gTgTCCCAGCCNgCKgTRTCRTC	10 pmol	S	9235–9260	64.1

Tm: temperature, Locked-Nucleic Acid bases are written as ‘ + _’, e.g., +A.

Degenerate bases: R = (A/G), W = (A/T), K = (T/G), Y = (C/T), N = (A/G/T/C).

^a S: sense orientation, AS: antisense orientation.

^b YFV strain; accession no: NC 002031.

^c Flavi all probe, ^d Flavi probe YFV, ^e Flavi probe DEN4.

Final Pan-Flavi assay comprise of Flavi all S, Flavi all S2 & Flavi all AS4 primer and Flavi all probe 3 mix.