Table 4.

Multiplex reference gene assay primer efficiency and variability.

	Infected tissue singleplex					Infected tissue multiplex
	Efficiency (%)	R-squared	Intra-assay variability (%)	Inter-assay variability (%)		Efficiency (%)	R-squared	Intra-assay variability (%)	Inter-assay variability (%)
Mouse (Mus musculus)					Mouse (Mus musculus)—Triplex
CCHFV	93.3	0.9991	0.26	0.55	CCHFV	101.5	0.9996	0.52	0.75
Gusb	99.1	0.9997	0.46	2.99	Gusb	103.9	0.9982	0.94	3.09
Ppia	97.8	0.9997	0.42	4.65	Ppia	101.0	0.9998	0.37	3.66
Pan.Ppia	103.0	0.9972	0.25	3.73	Pan.Ppia	104.5	0.9980	2.00	3.63
Hamster (Mesocricetus auratus)					Hamster (Mesocricetus auratus)—Duplex
NiV	90.7	0.9927	0.52	2.90	NiV	98.31	0.9994	0.84	1.79
Hrpt	94.0	0.9986	0.55	1.60	Hrpt	N/A	N/A	N/A	N/A
Ppia	98.1	0.9995	0.62	1.17	Ppia	98.80	0.9998	0.30	0.85
Pan.Ppia	94.3	0.9989	0.54	1.33	Pan.Ppia	95.30	0.9998	0.99	1.81
Guinea pig (Cavia porcellus)					Guinea pig (Cavia porcellus)—Triplex
LASV	90.6	0.9976	0.31	0.83	LASV	101.1	0.9999	0.76	1.87
Gapdh	94.3	0.9998	0.38	1.31	Gapdh	92.9	0.9967	2.33	3.08
Ppia	104.3	0.9917	0.41	3.44	Ppia	99.5	0.9999	0.29	1.28
Pan.Ppia	96.3	0.9996	0.5	2.50	Pan.Ppia	100.9	0.9865	4.47	4.18

Assay parameters were determined from standard curves generated by tenfold dilutions of 100 ng/µL total RNA isolated from spleens of CCHFV-infected mice, NiV-infected hamsters, or LASV-infected guinea pigs. Each assay was run as singleplex or was multiplexed (CCHFV and LASV: viral targets with species-specific RG and pan-rodent Ppia or species-specific Ppia; NiV: viral target with pan-rodent Ppia or species-specific Ppia). Intra- and inter-assay variability was assessed for both singleplex and multiplex assays using % CV.