A one-step multiplex qPCR assay for simultaneous identification and quantification of Leishmania martiniquensis and Leishmania orientalis/Leishmania chancei and detection and quantification of trypanosomatids in clinical samples

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Table 3

Repeatability and reproducibility analysis of the multiplex RT-qPCR assay.

DNA sample	DNA quantity (fg/reaction)	Intra assay					Inter assay
		Cq value^a			Mean ± SD^b	%CV^c	Cq value				Mean ± sd	%CV
		Replicate 1	Replicate 2	Replicate 3	Mean ± SD^b	%CV^c	Day 1	Day 2	Day 3	Day 4	Mean ± sd	%CV
L. martiniquensis	2 × 10⁵	12.88	12.78	12.89	12.85 ± 0.06	0.47	12.85	12.92	12.21	12.68	12.67 ± 0.32	2.50
	2 × 10³	20.60	20.69	20.62	20.64 ± 0.05	0.23	20.64	20.92	20.50	20.64	20.67 ± 0.18	0.85
	2 × 10⁰	32.28	32.56	32.64	32.49 ± 0.19	0.58	32.49	32.24	31.13	31.86	31.93 ± 0.59	1.86
L. orientalis	2 × 10⁵	15.15	15.35	15.55	15.35 ± 0.20	1.30	15.35	15.70	15.25	15.60	15.47 ± 0.21	1.35
	2 × 10³	23.34	22.94	23.22	23.17 ± 0.21	0.89	23.17	23.35	23.42	23.23	23.29 ± 0.11	0.49
	2 × 10⁰	33.21	33.77	33.62	33.53 ± 0.29	0.86	33.53	33.66	33.13	33.39	33.43 ± 0.23	0.69
Crithidia sp. (CLA-KP1)	2 × 10⁵	13.84	13.68	13.92	13.81 ± 0.12	0.88	13.81	14.05	14.20	14.02	14.02 ± 0.16	1.14
	2 × 10³	21.37	20.81	21.15	21.11 ± 0.28	1.34	21.11	21.12	20.98	21.23	21.11 ± 0.10	0.48
	2 × 10⁰	32.18	32.47	31.73	32.13 ± 0.37	1.16	32.13	32.27	32.19	32.17	32.19 ± 0.06	0.19

^aCq = Cycle threshold value; ^bMean ± SD = Mean ± standard deviation; ^c%CV = Coefficient of variation = (SD/mean) × 100.

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