Table 1.
Conventional PCR primer sequences and thermocycler conditions for assays used in this study
| Target gene | Size in base pairs (bp) | Primers names and sequence, reaction mix and run conditions |
|---|---|---|
|
Ehrlichia or Anaplasma16S rRNA |
420 bp |
GEPs 5’ CTG GCG GCA AGC YTA ACA CAT GCA AGT CGA ACG GA 3’ GEPr 5’ CTT CTT CTR TRG GTA CCG TCA TTA TCT TCC CYA YTG 3’ |
| |
|
For 25 μL mix: 12.5 μL MyTaq HS mix (Bioline), 0.2 μL 100 μM each primer (IDT® DNA Technology), 7.3 μl molecular grade water, + 5 μl template DNA. Denaturation 2 min @95°C, then 55 cycles 15 sec @94°C, annealing 10 sec @64°C, extension 15 sec @72°C, final extension 30 sec @72°C. |
|
Ehrlichia or Anaplasma16S rRNA |
973 bp |
GEPs 5’ CTG GCG GCA AGC YTA ACA CAT GCA AGT CGA ACG GA 3’ LongGEP1060r 5’CTG TGT RAG GTC CAG CCG AAC TGM SYC 3’ |
| |
|
As above except annealing and extension times extended to 25 and 30 sec |
|
Ehrlichia spp. sodb |
300 bp |
sodbF 5’- TTT AAT AAT GCT GGT CAA GTA TGG AAT CAT sodbR 5’- AAG CGT GTT CCC ATA CAT CCA TAG |
| |
|
For 50 μl mix: 24 μl MyTaq HS Mix (2X) (Bioline), 1 μl 50 uM each primer (Sigma-Aldrich), 4 μl molecular grade water + 20 μl template DNA. Single hot start cycle 3 min @95°C, then 55 cycles denaturation 10 sec @94°C, annealing 15 sec @58°C, extension 15 sec @72°C, then a single cycle 30 sec @72°C. |
|
E.chaffeensis
p28 |
590 bp |
EchP28F 5’- GAC CCA ACA GGT AGT GGT ATT AAC GG EchP28R 5’- CTG GGC TTA TAG AGT AGC TTA AAC CTA AC |
| |
|
For 25 μL mix: 12.5 μl MyTaq HS Mix (2X) (Bioline), 0.25 μl 50 uM each primer (Sigma-Aldrich), 7 μl molecular grade water + 5 μl template DNA. Single hot start cycle 3 min @95°C, then 55 cycles denaturation 15 sec @94°C, annealing 15 sec @58°C, extension 30 sec @72°C, then a single cycle 30 sec @72°C. |
|
E. ewingii
p28 |
215 bp |
EEM2F(Ref 32) 5′-GGA GCT AAA ATA GAA GAT AAT C EEM1R 5′-GTG CCA AAA GGT AAT ACA T |
| |
|
For 25 μl mix: 12.5 μl of MyTaq HS Mix (2X) (Bioline), 0.25 μl 50 μM each primer (Sigma-Aldrich), 2 μl molecular grade water + 10 μl template DNA. Single hot start cycle 3 min @95°C, then 55 cycles denaturation at 15 sec @94°C, annealing 15 sec @56°C, extension 30 sec @72°C, then a single cycle 1 min @72°C. |
|
A. platys
p44 |
520 bp |
Apl_p44F3 5’- GCT AAG TGG AGC GGT GGC GAT GA CAG Apl_p44R3 5’- CGA TCT CCG CCG CTT TCG TAT TCT TC |
| For 25 μL mix: 12.5 μl MyTaq HS Mix (2X) (Bioline), 0.3 μl 50 uM each primer (Sigma-Aldrich), 2 μl molecular grade water + 10 μl template DNA. Single hot start cycle 3 min @95°C, then 55 cycles denaturation 15 sec @94°C, annealing 10 sec @70°C, and extension 30 sec @72°C, then a single cycle 1 min @72°C. |