Table 2.
Primer sets for PCR amplification of FVBD agents
| Pathogen | Gene | Primers | Product size (bp) | PCR conditions | Reference |
|---|---|---|---|---|---|
|
Anaplasma spp./Ehrlichia spp. |
16S rRNA |
EHR16SD: 5′-GGT ACC YAC AGA AGA AGTCC-3′ |
345 |
95°C, 5 min; 35 cycles [94°C 30 sec, 55°C 30 sec, 72°C 90 sec]; 72°C, 5 min |
[12] |
| |
EHR16SR: 5′-TAG CAC TCA TCG TTT ACAGC-3′ |
||||
|
Babesia spp. |
18S rRNA |
PIRO-A: 5′-AAT ACC CAA TCC TGA CACAGG G-3′ |
400 |
95°C, 5 min; 35 cycles [94°C 30 sec, 55°C 30 sec, 72°C 90 sec]; 72°C, 5 min |
[13] |
| |
PIRO-B: 5′-TTA AAT ACG AAT GCC CCCAAC-3′ |
||||
|
Bartonella spp. |
16S-23S rRNA |
325 s: 5′-CTTCAGATGATGATCCCAAGCCTTCTGGCG-3′ |
500-800 |
95°C, 5 min; 55 cycles [95°C 15 sec, 66°C 15 sec, 72°C 15 sec]; 72°C, 1 min |
[14] |
| |
1100as: 5′-GAACCGACGACCCCCTGCTTGCAAAGCA-3′ |
||||
|
B. burgdorferi s.l. |
5S-23S rRNA |
Outer primers: |
380 |
|
[15] |
| |
23SN1: 5′-ACCATAGACTCTTATTACTTTGAC-3′ |
94.5°C, 1 min; 25 cycles [94°C 30 sec, 52°C 30 sec, 72°C 1 min]; 72°C, 5 min |
|||
| |
23SC1: 5′-TAAGCTGACTAATACTAATTACCC-3′ |
||||
| |
Inner primers: |
225 |
|
||
| |
23SN2: 5′-ACCATAGACTCTTATTACTTTGACCA-3′ |
94.5°C, 1 min; 40 cycles [94°C 30 sec, 55°C 30 sec, 72°C 1 min]; 72°C, 5 min |
|||
| |
5SCB: 5′-biotin-GAGAGTAGGTTATTGCCAGGG-3′ |
||||
|
Hepatozoon spp. |
18S rRNA |
HEP-F: 5′-ATA CAT GAG CAA AAT CTC AAC-3′ |
626-666 |
95 °C, 5 min; 34 cycles [95 °C 20 sec, 55 °C, 30 sec, 72 °C, 90 sec]; 72 °C, 5 min |
[16] |
| |
HEP-R: 5′-CTT ATT ATT CCA TGC TGC AG-3′ |
||||
|
Leishmania spp. |
Small subunit rRNA |
Outer primers: |
603 |
|
[17] |
| |
R221: 5′-GGTTCCTTTCCTGATTTACG-3′ |
94°C, 5 min; 35 cycles [94°C 30 sec, 60°C 30 sec, 72°C 30 sec]; 72°C, 10 min |
|||
| |
R332: 5′-GGCCGGTAAAGGCCGAATAG-3′ |
||||
| |
Inner primers: |
358 |
|
[18] |
|
| |
R223: 5′-TCCCATCGCAACCTCGGTT-3′ |
94°C, 5 min; 35 cycles [94°C 30 sec, 65°C 30 sec, 72°C 30 sec]; 72°C, 10 min |
|||
| R333: 5′-AAAGCGGGCGCGGTGCTG-3′ |