Pathogen | Primer sequences (5'–3') | Amplicon length (bp) | PCR cyclera | Mastermix | Protocol | Reference primer sequences |
---|---|---|---|---|---|---|
A. phagocytophilum | locus: major surface protein 2 (msp2) gene (forward: ATG GAA GGT AGT GTT GGT TAT GGT ATT; reverse: TTG GTC TTG AAG CGC TCG TA; probe: FAM-TGG TGC CAG GGT TGA GCT TGA GAT TG-BHQ1) | 75 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation/polymerase activation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [108] |
Babesia spp. | locus: 18S ribosomal RNA gene (forward: TGA ACG AGG AAT GCC TAG TATG; reverse: CCG AAT AAT TCA CCG GAT CAC TC; probe: FAM-AAG TCA TCA GCT TGT GCA GAT TAC GTC CCT-BHQ1) | 116 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation/polymerase activation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [65] |
B. microti | locus: 18S ribosomal RNA gene (forward: CAG GGA GGT AGT GAC AAG AAA TAA CA; reverse: GGT TTA GAT TCC CAT CAT TCC AAT; probe: FAM-TAC AGG GCT TAA AGT CT-MGBNFQ) | 71 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [109] |
Borrelia spp. | locus: 16S ribosomal RNA gene (forward: GGTCAAGACTGACGCTGAGTCA; reverse: GCGGCACACTTAACACGTTAG; probe: FAM-TCT ACG CTG TAA ACG ATG CAC ACT TGG TG-BHQ1) | 136 | Roche LightCycler 96 | TaqMan Fast Advanced Master Mix (final primer conc.: 0.4 μM; final probe conc.: 0.25 μM; reaction volume: 25 μl; sample volume: 5 μl) | Uracil-N glycosylase incubation (50 °C, 120 s); initial denaturation/polymerase activation (95 °C, 20 s); 2-step-amplification (45× 95 °C, 3 s; 60 °C, 30 s) | [110] |
B. miyamotoi | locus: 16S ribosomal RNA gene (forward: CGC TGT AAA CGA TGC ACA CTT GGT GTT AAT C; reverse: CGG CAG TCT CGT CTG AGT CCC CAT CT;probe: FAM-CCT GGG GAG TAT GTT CGC AAG AAT GAA ACT C-BHQ1) | 352 | Roche LightCycler 96 | TaqMan Fast Advanced Master Mix (final primer conc.: 0.4 μM; final probe conc.: 0.25 μM; reaction volume: 25 μl; sample volume: 5 μl) | Uracil-N glycosylase incubation (50 °C, 120 s); initial denaturation/polymerase activation (95 °C, 20 s); 2-step-amplification (45× 95 °C, 3 s; 60 °C, 30 s) | [19] |
"Ca. Neoehrlichia mikurensis" | locus: 16S ribosomal RNA gene (forward: ATC CTG GCT CAG AAC GAA CG; reverse: TGA TCG TCC TCT CAG ACC AGC; probe: FAM-ACC CAT AGT AAA CTA CAG CTA CA-MGBNFQ) | 280 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation/polymerase activation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [99] |
Rickettsia spp. | locus: citrate synthase-encoding gene (gltA) (forward: TCG CAA ATG TTC ACG GTA CTT T; reverse: TCG TGC ATT TCT TTC CAT TGT G; probe: FAM-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-BHQ1) | 74 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation/polymerase activation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [26] |
R. helvetica | locus: 23S ribosomal RNA gene (forward: TTT GAA GGA GAC ACG GAA CAC A; reverse: TCC GGT ACT CAA ATC CTC ACG TA; probe: FAM-AAC CGT AGC GTA CAC TTA-MGBNFQ) | 65 | AB QuantStudio 12K Flex | LightCycler Multiplex DNA Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Initial denaturation/polymerase activation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [26] |
TBEV | locus: envelope gene (forward: GGT TTG TGA GGC AAA AAA GAA; reverse: TCC CGT GTG TGG TTC GAC TT; probe: FAM-AAG CCA CAG GAC ATG TGT ACG ACG CC-BHQ1) | 88 | AB QuantStudio 12K Flex | LightCycler Multiplex RNA Virus Master & 50 nM external ROX (final primer conc.: 0.5 μM; final probe conc.: 0.25 μM; reaction volume: 10 μl; sample volume: 2.5 μl) | Reverse transcription (50 °C, 600 s); initial denaturation (95 °C, 30 s); 2-step-amplification (45× 95 °C, 5 s; 60 °C, 30 s); cooling (40 °C, 30 s) | [10] |