Table 1. Primer sequences used for RT-RPA and RT-qPCR assays.

Primer Name	Sequence (5′ → 3′)	Nucleotidea	Source
JJV2Fb	CAAGAGTCAATGTTTAGGTGGATGAG	5003	Jothikumar et al.14
COG2Rb	TCGACGCCATCTTCATTCACA	5101	Jothikumar et al.14
Ring2-TPb	TGGGAGGGCGATCGCAATCT	5048	Jothikumar et al.14
NOF5	CCACGGCCCAGCATTTTACAGCAAAATCAGC	4918	This Paper
NOF7	CCATACAATTGATGTCCCTACTGGGGGAGGCCGC	4880	This Paper
NOR9	TTCTAGGGGATACTGTAAACTCTCCACCAGGGGC	5292	This Paper
G2R10	CCTGGGGCATTTCTAGGGGATACTGTAAACTCTCC	5304	This Paper
NOR11	CTACGGGCTCCAAAGCCATAACCTCATTGTTGACC	5182	This Paper
NOR12	CCAAAGCCATAACCTCATTGTTGACCTCTGGG	5172	This Paper
NOP1d	ATTTTTACGTGCCCAGACAAGAGCCAATGT3CAHA1GGATGAGATTCTCAGA	4987	This Paper
T7GII.4Fc	TAATACGACTCAACTATAGCAAGAGTCAATGTTTAGGTGGATGAG	5003	This Paper
GII.4R2c	GTTGGGAAATTCGGTGGGACTG	5182	This Paper

RT-PCR and RT-qPCR reactions were both cycled at a 15 min reverse transcription cycle at 50 °C, followed by reverse transcriptase inactivation at 95 °C for 2 min, then amplification for 30 or 45 cycles of 95 °C for 15 sec, 55 °C or 54 °C for 30 sec, and 72 °C for 30 sec, respectively. Primer and probe reaction concentrations were all 200 nM.

^aNucleotide corresponding to 5′ of primer on GII.4 New Orleans sequence (GenBank JN595867.1).

^b54 °C annealing temperature and 2.1 pmol primer in 50 μl reaction used for RT-qPCR primers and probe.

^c55 °C annealing temperature and 2.1 pmol primer in 50 μl reaction used for RT-PCR amplification of standard.

^dFor probe modifications: 3 = internal dT-FAM; H = THF; 1 = internal dT-BHQ1. Probe has 3′ C3-spacer for blocking extension.