Table 2.

 Results of Evaluation of Urine Preparation and Storage Conditions for the Detection of JEV by RT-qPCR

		JEV RT-qPCR Results, Cq Value
		140 µL							5 mL, Microcentrifuge
		Extract 1		Extract 2		Extract 3			Extract 1		Extract 2		Extract 3
		1	2	1	2	1	2	Mean	1	2	1	2	1	2	Mean
Baseline, t0
Neat	High	27.49	ND	27.56	ND	27.29	ND	27.45
	Medium	31.10	ND	31.47	ND	31.22	ND	31.27
	Low	35.51	ND	35.27	ND	33.85	ND	34.87
Stored for 6 h at 4°C
Neat	High	30.25	30.7	31.02	30.67	30.29	33.14	31.01	30.64	30.01	29.98	30.11	30.26	30.1	30.18
	Medium	34	35.6	35.92	34.95	35.3	34.05	34.97	32.64	34.68	35.09	33.52	33.85	34.83	34.1
	Low	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
Stored for 4 d at –80°C
Neat	High	27.95	28.03	29.57	28.98	29.08	27.68	28.55	31.73	30.7	31.61	30.31	ND	ND	31.08
	Medium	32.62	31.85	33.38	32.47	32.1	30.25	32.11	31.71	31.68	33.71	32.18	ND	ND	32.32
	Low	Neg	37.78	36.07	33.75	38.6	39.32	Nega	Neg	Neg	Neg	Neg	ND	ND	Neg
With buffer AVL	High	27.17	27.46	26.79	26.69	26.36	26.84	26.88	25.95	25.92	26.06	25.75	ND	ND	25.92
	Medium	30.83	30.6	31.02	30.19	30.32	30.04	30.50	28.86	28.44	28.99	29	ND	ND	28.83
	Low	36.24	34.11	35.35	36.4	34.18	35.23	35.25	33.33	33.42	33.36	33.78	ND	ND	33.47
Stored for 14 d at –80°C
Neat	High	32.03	31.78	28.41	29.07	28.46	28.59	29.72
	Medium	32.78	32.67	34.63	35.75	33.25	34.11	33.87
	Low	Neg	Neg	Neg	38.22	Neg	Neg	Nega
With buffer AVL	High	26.91	26.62	26.56	26.74	26.88	26.86	26.76
	Medium	30.93	31.1	30.03	29.74	29.87	29.9	30.26
	Low	35.1	34.62	35.58	34.4	33.79	33.9	34.56
Stored for 30 d at –80°C
Neat	High	32.13	ND	32.76	ND	ND	ND	32.44
	Medium	37.16	ND	36.03	ND	35.13	ND	36.10
	Low	Neg	ND	Neg	ND	38.70	ND	Nega
With buffer AVL	High	30.76	ND	30.38	ND	30.85	ND	30.66
	Medium	30.08	ND	34.25	ND	33.65	ND	32.66
	Low	39.13	ND	37.66	ND	37.54	ND	38.11

“T0” indicates extracted and tested immediately after virus spiking and preparation of dilutions. Three extraction replicates were performed, on the same run, and 2 RT-qPCR replicates for each extraction. “Controls” indicates PCR-positive controls (x2 JEV G3) and negative controls (no template) in duplicate positive and negative, respectively, MS2 extraction positive control positive for each sample. “ND” indicates not done. This was in an effort to conserve reagents and microcentrifuge kits. Cq >40 were reported as negative and ≤40 as positive.

Abbreviations: JEV, Japanese encephalitis virus; RT-qPCR, reverse transcription real-time polymerase chain reaction.

^aMean reported as negative if 1 or more extracts were negative.