. 2018 Mar 23;13(3):e0194412. doi: 10.1371/journal.pone.0194412

Table 5. Cq results for validation of the optimised JEV RT-qPCR assays.

Assay	JEV Strain	Day	N	10⁻³			10⁻⁴			10⁻⁵			10⁻⁶			10⁻⁷			10⁻⁸			10⁻⁹			10⁻¹⁰			10⁻¹¹
Assay	JEV Strain	Day	N	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD	Cq	Mean	SD
Pyke	G1	1	1	21.30	21.16	0.14	24.45	24.44	0.05	27.66	27.65	0.05	$31.14$	$31.06$	$0.07$	39.28	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-
Pyke	G1	1	2	21.17			24.39			27.69			$31.03$			35.24			No Cq			No Cq			No Cq
Pyke	G1	1	3	21.02			24.49			27.60			$31.02$			No Cq			No Cq			No Cq			No Cq
Pyke	G1	2	1	21.00	21.05	0.05	24.20	24.22	0.04	27.19	27.27	0.07	$30.55$	$30.71$	$0.14$	34.27	35.95	2.52	No Cq	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-
Pyke	G1	2	2	21.05			24.27			27.30			$30.75$			38.84			No Cq			No Cq			No Cq
Pyke	G1	2	3	21.09			24.19			27.32			$30.82$			34.73			No Cq			No Cq			No Cq
NS2A	G1	1	1	20.57	20.47	0.18	24.00	23.96	0.10	27.10	27.21	0.10	30.58	30.62	0.09	$34.03$	$34.36$	$0.29$	36.98	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-
NS2A	G1	1	2	20.57			23.85			27.29			30.72			$34.51$			No Cq			No Cq			No Cq
NS2A	G1	1	3	20.26			24.04			27.24			30.56			$34.54$			36.27			No Cq			No Cq
NS2A	G1	2	1	20.44	20.33	0.12	23.46	23.60	0.13	26.91	26.84	0.06	30.09	30.06	0.18	$33.58$	$33.57$	$0.19$	39.83	37.60	1.99	No Cq	No Cq	-	No Cq	No Cq	-
NS2A	G1	2	2	20.33			23.71			26.82			29.87			$33.37$			36.93			No Cq			No Cq
NS2A	G1	2	3	20.21			23.64			26.80			30.22			$33.75$			36.03			No Cq			No Cq
NS2A	G3	1	1													30.51	30.65	0.20	$34.06$	$34.18$	$0.17$	No Cq	No Cq	-	No Cq	No Cq	-
NS2A	G3	1	2													30.79	30.65	0.20	$34.30$	$34.18$	$0.17$	39.67	No Cq	-	No Cq	No Cq	-
NS3	G3	1	1				22.46	22.66	0.28	25.96	26.27	0.27	28.78	28.81	0.38	31.85	31.87	0.04	$34.57$	$35.63$	$1.43$	38.49	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-
NS3	G3	1	2				22.98			26.42			28.45			31.84			$37.25$			38.25			No Cq			No Cq
NS3	G3	1	3				22.55			26.42			29.20			31.92			$35.07$			No Cq			No Cq			No Cq
NS3	G3	2	1				22.95	22.78	0.21	26.08	26.04	0.09	29.72	29.77	0.13	31.72	32.01	0.25	$35.22$	$35.30$	$0.10$	39.85	No Cq	-	No Cq	No Cq	-	No Cq	No Cq	-
NS3	G3	2	2				22.85			25.94			29.68			32.18			$35.27$			No Cq			No Cq			No Cq
NS3	G3	2	2				22.54			26.10			29.92			32.12			$35.41$			No Cq			No Cq			No Cq

Dilution series performed using JEV G1 RNA (JEV/CNS769/Laos/2009; 1.3x10⁶ RNA copies/μl; GenBank: KC196115, EVA 001V-02217) and JEV G3 RNA (UVE/JEV/UNK/TW/RP9-190; 1.2 x10⁷ RNA copies/μl; GenBank KF907505, EVA 001V-02344) with triplicates on 2 different days. N = replicate number. LOD (highest dilution found positive by all replicates) is shaded in grey. RT-qPCR performed with Fastvirus kit (TaqMan® Fast Virus 1-Step) with a reaction volume of 50μL, sample volume of 30μl, and primer and probe concentrations of 600nM and 300nM respectively. Thermocycling conditions were 50°C for 5 minutes, 95°C for 20 seconds and 45 x (95°C for 15 seconds + x°C for 60 seconds). The optimal annealing temperature ‘x°C’ was different for each assay: 62°C, 60°C and 56°C for the Pyke, NS2A and NS3 assays respectively.