Table 1.

Detection limit and Ct value comparison of the multiplex real-time polymerase chain reaction (PCR) assays on diluted bacteria-spiked PBS and whole blood

CFU number/reaction
			104	103	102	10	1	R2	Slope	E	Intercept	P-value
S. Typhi (FAM)	PBS	Ct value*	23.6	27.27	30.65	33.20	(35.84)	0.992	−3.219	104.5	36.73	0.347
		CV (%)	0.24	0.26	0.93	0.17	(1.08)
	Blood	Ct value	23.31	26.64	30.07	32.94	(35.86)	0.988	−3.234	103.8	38.31
		CV (%)	0.42	1.88	0.93	1.54	(0.80)
S. Paratyphi A (VIC)	PBS	Ct value*	24.77	28.37	31.53	34.32	Neg	0.989	−3.208	105.1	37.79	1
		CV (%)	0.12	0.56	0.18	2.49	/
	Blood	Ct value*	24.19	27.35	31.4	34.68	Neg	0.989	−3.567	90.7	38.31
		CV (%)	0.41	1.64	1.54	1.13	/
S. Typhimurium (Cy5)	PBS	Ct value*	23.92	27.45	30.71	33.66	(36.68)	0.996	−3.248	103.2	37.06	0.272
		CV (%)	0.06	0.26	0.36	1.13	/
	Blood	Ct value	24.80	28.29	31.49	34.23	(35.75)	0.982	−3.149	107.7	37.57
		CV (%)	2.02	0.94	1.36	1.77	(1.68)

Ct = cycle threshold; CV = coefficient of variance. R 3.4.4 software was used for calculation of adjusted R-square, slope, and efficacy through a simple linear regression model and for P-value determination (nonparametric Wilcoxon’s test).

* Mean Ct value from three individual replicates. Intra-assay variation (CV [%)]) was calculated by measuring the coefficient of variance of the Ct value on at least three individual assays. R² = coefficient of determination; Slope = slope of the curve; E = amplification efficiency (%); E = ([10−^1/slope] − 1) × 100. Intercept = mean of the Ct value when CFU number/reaction = 0. Data in () are not considered for final analysis in the absence of full detection for replicates.