Table 2.

Description of primers, amplicon sizes and thermal sequences used in conventional and nested PCR assays for Ehrlichia spp. and Anaplasma spp. Protocols marked with * represent nested PCR assays.

Agents	Primers sequences	Size (bp)	Thermal sequences	References
Anaplasma spp. (rrs gene)* External primers  gE3a  gE10R Internal primers  gE2  gE9f	5′-CACATGCAAGTCGAACGGATTATTC-3′ 5′-TTCCGTTAAGAAGGATCTAATCTCC′-3′ 5′-GGCAGTATTAAAAGCAGCTCCAGG-3′ 5′-AACGGATTATTCTTTATAGCTTGCT-3′	932 546	94 °C for 5 min 40 cycles: 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min 72 °C for 5 min	30
Ehrlichia spp. (dsb gene)  dsb-330 (F)  dsb-728 (R)	5′- GATGATGTCTGAAGATATGAAACAAAT-3′ 5′-CTGCTCGTCTATTTTACTTCTTAAAGT-3′	409	95 °C for 2 min; 50 cycles: 95 °C for 15 s, 58 °C for 30 s and 72 °C por 30 segundos 72 °C for 5 min	34
Anaplasma spp. (ITS—23S–5S)  ITS2F  ITS2R	5′-AGGATCTGACTCTAGTAACGAG-3′ 5′-CTCCCATGTCTTAAGACAAAG-3′	300	94 °C for 2 min, 35 cycles; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min 72 °C for 5 min	33
Ehrlichia spp. (groEL gene)  groEL124-F1  groEL808-R1	5′-ATTAAGCCAGAAGAACCATTAGC-3′ 5′-TACTGCAATATCACCAAGCATATC-3′	680	95 °C for 5 min, 40 cycles: 95 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s 72 °C for 5 min	36
Ehrlichia spp. (sodB gene)  sodbEhr1600-F  sodbEhrl600-R	5′-ATGTTTACTTTACCTGAACTTCCATATC-3′ 5′-ATCTTTGAGCTGCAAAATCCCAATT-3′	600	94 °C for 3 min; 55 cycles: 94 °C for 10 s; 58 °C for 10 s; 72 °C por 15 s 72 °C for 30 s 72 °C for 5 min	37
Ehrlichia spp. (omp-1 gene)* External primers  conP28-F1  conP28-R1 Internal primers  conP28-F2  conP29-R2	5′AT(C/T)AGT(G/C)AAA(A/G)TA(T/C)(A/G)T(G/A)CCAA-3′ 5′TTA(G/A)AA(A/G)G(C/T)AAA(C/T)CT(T/G)CCTCC-3′ 5′CAATGG(A/G)(T/A)GG(T/C)CC(A/C)AGA(AG)TAG-3′ 5′TTCC(T/C)TG(A/G)TA(A/G)G(A/C)AA(T/G)TTTAGG-3′	300	94 °C for 3 min, 35 cycles: 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min 72 °C for 5 min	38