Skip to main content

Table 1 Conventional PCR primer sequences and thermocycler conditions for assays used in this study

From: Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members

Target gene Size in base pairs (bp) Primers names and sequence, reaction mix and run conditions
Ehrlichia or Anaplasma 16S 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 Anaplasma 16S 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.