Table 4.

Average Ct values obtained when the EEEV, SLEV and GFP real-time RT-PCR assays were performed on extracts of cerebrospinal fluid specimens spiked with SLEV culture and EEEV transcript.

Sample	Virus/transcript spike	Average Ct for viral assay	Ct range for viral assay	CV for viral assay	Average Ct for GFP assay	Ct range for GFP assay	CV for GFP assay
NTC	–	≥45	≥45 – ≥45	0	≥45	≥45 – ≥45	0
EEEV spike
	Negative	≥45	≥45 – ≥45	0	35.01	34.89 – 35.11	0.2%
	Low	39.22	37.55 – 41.3	2.85%	35.30	34.87 – 35.87	0.97%
	Medium	36.41	35.44 – 38.81	2.47%	35.65	34.92 – 38.66	3.04%
	High	32.21	31.57 – 32.62	0.83%	35.52	34.92 – 35.97	1.09%
SLEV spike
	Negative	≥45	≥45 – ≥45	0	35.21	34.87 – 35.87	0.98%
	Low	39.62	38.04 – 41.75	2.44%	35.27	34.92 – 35.97	1.09%
	Medium	34.07	33.48 – 34.78	0.75%	35.27	34.96 – 35.69	0.77%
	High	29.67	29.39 – 30.08	0.63%	35.73	34.95 – 38.66	3.02%

NTC, no template control; the high, medium and low spikes for SLEV were 9000, 90, 9 gene copies/reaction respectively; the high, medium and low spikes for EEEV were 5000, 500, 50 gene copies/reaction respectively. The average numbers are from 10 assays performed in duplicate for virus detection and performed singly for GFP detection.