Table 1

Nested and real-time PCR assays

TargetAssayPrimer/probe namePrimer/probe sequence (5′ → 3′)cReference(s)
Rickettsia sp.17 kDa primaryaR17-122CAG AGT GCT ATG AAC AAA CAA GG5, 6
R17-500CTT GCC ATT GCC CAT CAG GTT G
Spotted fever group17 kDa nestedaTZ15TTC TCA ATT CGG TAA GGG C7
TZ16ATA TTG ACC AGT GCT ATT TC
Typhus group17 kDa nestedaRP2TTC ACG GCA ATA TTG ACC TGT ACT GTT CC6
RPIDCGG TAC ACT TCT TGG TGG CGC AGG AGG T
Spotted fever groupOmpA primaryaRR190-70ATG GCG AAT ATT TCT CCA AAA12, 13
RR190-701GTT CCG TTA ATG GCA GCA TCT
OmpA nesteda190-FN1AAG CAA TAC AAC AAG GTC12, 13
190-RN1TGA CAG TTA TTA TAC CTC
OmpA seminestedaRR190-70ATG GCG AAT ATT TCT CCA AAA8, 13
RR190-602AGT GCA GCA TTC GCT CCC CCT
Rickettsia sp.Pan-Rickettsia real-time PCRbPanR8_FAGC TTG CTT TTG GAT CAT TTG GThis article
PanR8_RTTC CTT GCC TTT TCA TAC ATC TAG T
PanR8_PFl-CCT GCT TCT ATT TGT CTT GCA GTA ACA CGC CA-BHQ1
R. rickettsiiR. rickettsii real-time PCRbRRi6_FAAA TCA ACG GAA GAG CAA AACThis article
RRi6_RCCC TCC ACT ACC TGC ATC AT
RRi6_PcFl-TCC TCT CCA ATC AGC GAT TC-BHQ1
  • a PCR conditions were as described by Paddock et al., 2004 (11).

  • b Each 25-μl reaction mixture contained 1× PerfeCTa Multiplex qPCR SuperMix (Quanta, Maryland) with 5 μl DNA template, 1 μM (each) forward and reverse primers, and 40 nM probe concentration; 8 min at 95°C and then 45 cycles of 5 s at 95°C and 30 s at 60°C. The pan-Rickettsia and R. rickettsii real-time PCR assays target the 23S rRNA and a gene encoding hypothetical protein A1G_04230 (GenBank accession no. ABV76353; R. rickettsii Sheila Smith strain), respectively.

  • c Probe labels are fluorescein (Fl) and black hole quencher 1 (BHQ1).