Comparison of seven different RT-qPCR for diagnostic of Foot-and-Mouth disease virus

Fonseca Junior, Antonio Augusto; Laguardia-Nascimento, Mateus; Ferreira, Amanda Petrina Scotá; Silva, René Ribeiro da; Rivetti Júnior, Anselmo Vasconcelos; Camargos, Marcelo Fernandes

doi:10.1590/1809-6891v25e-76304E

Antonio Augusto Fonseca Junior^**corresponding author: antonio.fonseca@agro.gov.br

PCR Standardization

Repeatability

Sensitivity

Specificity

Manuscript Preparation

Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA/MG), Pedro Leopoldo, Minas Gerais, Brazil

https://orcid.org/0000-0002-2962-5616

Mateus Laguardia-Nascimento

Nucleic Acid Extraction

Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA/MG), Pedro Leopoldo, Minas Gerais, Brazil

https://orcid.org/0000-0003-4428-0764

Amanda Petrina Scotá Ferreira

Nucleic Acid Extraction

Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA/MG), Pedro Leopoldo, Minas Gerais, Brazil

https://orcid.org/0000-0002-4105-8052

René Ribeiro da Silva

Viral Isolation

Laboratório Federal de Defesa Agropecuária do Pará, Belém, Pará, Brazil

https://orcid.org/0000-0002-3943-3757

Anselmo Vasconcelos Rivetti Júnior

Manuscript Preparation

Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA/MG), Pedro Leopoldo, Minas Gerais, Brazil

https://orcid.org/0000-0003-2988-2652

Marcelo Fernandes Camargos

Manuscript Preparation

Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA/MG), Pedro Leopoldo, Minas Gerais, Brazil

https://orcid.org/0000-0001-8457-7875

*corresponding author: antonio.fonseca@agro.gov.br

Conflict of Interest

There are no conflicts of interest to declare.

RT-qPCR	Oligonucleotide	Sequence	PCR Mix	Reference
FMDV.3D.107	FMDV.3D.107.F	ACTGGGTTTTACAAACCTGTGA	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.4 µM, Probe 0.2 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(7)
	FMDV.3D.107.R	GCGAGTCCTGCCACGGA
	FMDV.3D.107.S	FAM-TCCTT TGCAC GCCGT GGGAC-BHQ1
FMDV.3D.99	FMDV.3D.99.F1	ACTGGGTTTTACAAACCTGTGATG	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.4 µM, Probe 0.2 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(24)
	FMDV.3D.99.F2	CTGGGTTTTATAAACCTGTGATGGC
	FMDV.3D.99.R1	CCACGGAGATCAACTTCTCCT
	FMDV.3D.99.R2	TGCCACAGAGATCAACTTCTCC
	FMDV.3D.99.R3	CCACGGAAATCAACTTCTCCTG
	FMDV.3D.99.S	FAM-TCTCCTTTGCAC-GCCGTGG-BHQ1
FMDV.3D.130	FMDV.3D.130.F	GGACCATACAGGAGAAGTTGA	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.4 µM, Probe 0.2 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(25)
	FMDV.3D.130.R	CGCAGGTAAAGTGATCTGTAGC
	FMDV.3D.130.S	FAM-CTCCGTGGCAG-GACTCGCAGT-BHQ1
FMDV.3D.183	FMDV.3D.183.F FMDV.3D.183.R	GAC AAA GGT TTT GTT CTT GGT CA TGCGAGTCCTGCCACGGA	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.6 µM, Probe 0.3 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(26)
	FMDV.3D.183.S	FAM-ATC CTC TCC TTT GCA CGC CGT GGG ACC AT-BHQ1		(26)
FMDV.IRES.145	FMDV.IRES.145.F	TAA CAW GGA CCC RCS GGG CC	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.4 µM, Probe 0.2 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(27)
	FMDV.IRES.145.R	TGA AGG GCA TCC TTA GCC TG
	FMDV.IRES.145.S	FAM - CAT GTG TGC AAY CCC AGC ACR G - BHQ
FMDV.3D.88	FMDV.3D.88.F	ACTGGGTTTTAWAAACCTGTGATG	12.5 µL QuantiNova RT-qPCR (Qiagen, Germany), Primers 0.4 µM, Probe 0.2 µM, 3 µL DNA sample, water q.s. 25 µL (1) 95°C 5 minutes, (45) 95°C 10 seconds, 60 °C 60 seconds	(17)
	FMDV.3D.88.R	TCAACTTCTCCTGKATGGTCCCA
	FMDV.3D.88.S	FAM-ATCCTCTCCTTTG-CACGC-Iowa Black

		Plasmid		FMDV A		FMDV C		FMDV O		FMDV SAT
RTqPCR	Effciency	Last Positive Dilution^* * Last positive dilution and number of positive replicates.	Median Ct^ Median Ct and standard deviation	Last Positive Dilution	Median Ct	Last Positive Dilution	Median Ct	Last Positive Dilution	Median Ct	Last Positive Dilution	Median Ct
FMDV.3D.107	96.2%	-5 x3	39.47 (0.54)	-7 x2	40.56 (0.3)	-8 x2	40.59 (0.83)	-5 x2	39.67	-6 x2	41.06 (0.21)
FMDV.3D.88	98.7%	-5 x3	39.2 (0.34)	-7 x2	40.1 (0.8)	-7 x2	43.48 (2.46)	-6 x3	x35.99 (0.30)	-6 x3	39.75 (0.42)
FMDV.3D.99	99%	-5 x2	37.91 (1,2)	-7 x2	39.41 (0.96)	-8 x3	38.51 (2.23)	-5 x3	37.86	-7 x1	37,89
FMDV.3D.130	93%	-5 x3	37.94 (0.83)	-7 x3	37.88 (0.794)	-8 x3	39,18 (0.34)	-5 x3	39.34	-6 x1	38.81
FMDV.3D.183	91.2%	-7 x3	39.78 (0.71)	-7 x3	36.31 (1.03)	-7 x3	36.92 (0+95)	-5 x1	37.64	-6 x1	36.57

Sample^* * Samples from oubreaks (collected tissues/samples with diagnose confirmed by viral isolation) were provided by Lanagro/PA	FMDV.3D.107	FMDV.3D.88	FMDV.3D.99	FMDV.3D.130	FMDV.3D.183
1	26.18	26.70	25.23	26.71	25.79
2	32.03	33.04	29.72	31.04	31.06
3	35.03	35.60	32.72	34.14	34.07
4	32.79	33.05	30.92	32.12	32.11
5	27.27	27.88	25.67	26.24	26.37
6	28.30	29.10	27.02	27.96	27.76
7	26.78	26.78	25.10	26.34	25.73
8	22.72	23.31	21.13	23.25	22.13
9	23.35	23.64	21.62	24.54	22.66
10	24.38	24.91	22.89	24.51	24.79
11	22.12	23.72	20.89	23.37	22.20
12	19.92	20.33	19.23	21.79	18.31
13	24.34	24.54	23.64	26.33	24.81
14	38.50	-	38.14	-	37.38
15	38.97	39.20	38.03	-	37.38
16	40.19	40.64	39.07	39.30	39.64

RT-qPCR	Repeatability	Error of Measure	Uncertainty of Measure	Repeatability vs FMDV.3D.107
FMDV.3D.107	0.72	1.34	0.33	-
FMDV.3D.88	0.85	1.40	0.44	0.69
FMDV.3D.99	0.43	1.37	0.28	0.57
FMDV.3D.130	1.06	2.01	0.49	0.93
FMDV.3D.183	0.61	1.42	0.38	0.66

Sample	FMDV.3D.107	FMDV.3D.88	FMDV.3D.99	FMDV.3D.130	FMDV.3D.183
1	18.79	18.20	16.45	20.21	17.71
2	19.28	20.67	16.67	20.09	18.87
3	20.76	21.20	19.27	22.96	20.25
4	26.56	26.90	24.19	28.32	26.88
5	36.98	37.50	34.02	39.39	36.53
6	36.41	36.88	34.92	36.56	36.06
7	39.47	-	38.87	-	45.08
8	-	-	38.94	-	40.50
9	-	-	37.43	-	-
10	-	-	-	-	-
11	40.52	-	38.90	-	-
12	37.76	38.53	36.13	37.86	38.74
13	-	-	-	-	-
14	-	42.00	-	-	42.00
15	-	-	-	-	-
16	27.29	27.94	26.01	26.69	26.01
17	27.06	27.55	25.77	27.27	26.16
18	-	-	-	-	-

[1] *corresponding author: antonio.fonseca@agro.gov.br

Brasil

Brasil

Comparison of seven different RT-qPCR for diagnostic of Foot-and-Mouth disease virus

Abstract