Table 1: Analytical sensitivity of the multiplexed RT-qPCR assay to screen for variants of concern using primer/probe sets targeting key deletions.

Listed are cycle threshold (Ct) values for the three primer-probe sets targeting the SARS-CoV-2 nucleocapsid (N1-FAM), ORF1a 3675-3766 deletion (ORF1a-Cy5), and spike 69-70 deletion (Spike-HEX). Two-fold dilutions of synthetic control RNA (Wuhan-Hu-1 and B.1.1.7) were tested in triplicate.

RNA	Concentration	N1-FAM			ORF1a-Cy5			Spike-HEX
Wuhan-Hu-1	100 copies/μL	30.4	30.5	30.4	31.1	31.2	31.1	30.8	30.8	30.9
	50 copies/μL	31.5	31.5	31.4	32.1	32.1	32.0	31.9	31.6	31.7
	25 copies/μL	32.3	32.7	32.7	33.0	33.1	33.2	32.8	32.8	33.0
	12 copies/μL	33.3	33.7	33.3	34.1	34.2	34.2	34.0	33.6	34.1
	6 copies/μL	34.5	34.3	34.9	35.1	35.2	35.4	35.1	34.3	35.6
	3 copies/μL	35.0	37.0	36.1	35.6	36.6	36.8	35.4	36.1	35.8
	1 copy/μL	37.0	37.0	35.7	37.2	36.7	37.0	36.4	36.4	36.3
B.1.1.7	100 copies/μL	29.1	29.2	29.2	ND	ND	ND	ND	ND	ND
	50 copies/μL	30.1	30.2	30.2	ND	ND	ND	ND	ND	ND
	25 copies/μL	31.1	31.2	31.2	ND	ND	ND	ND	ND	ND
	12 copies/μL	32.5	32.2	32.1	ND	ND	ND	ND	ND	ND
	6 copies/μL	33.0	33.2	33.1	ND	ND	ND	ND	ND	ND
	3 copies/μL	33.7	34.6	34.0	ND	ND	ND	ND	ND	ND
	1 copy/μL	35.0	35.2	35.3	ND	ND	ND	ND	ND	ND

Wuhan-Hu-1 = Twist synthetic RNA control 2; B.1.1.7 = Twist synthetic RNA control 14; N1-FAM = CDC N1 primer-probe set targeting the nucleocapsid with FAM fluorophore¹⁶; ORF1a-Cy5 = Yale primer-probe set targeting the ORF1a gene 3675-3677 deletion with Cy5 fluorophore⁸; Spike-HEX = Yale primer-probe set targeting the spike gene 69-70 deletion with HEX fluorophore⁸.