Table 1 PCR methods, target genes, primers, expected PCR product sizes, and reaction and amplification conditions as well as references used for detection of tick-borne pathogens

Hepatozoon spp.

Conventional PCR

18S rDNA

5′-ATACATGAGCAAAATCTCAAC-3′

5′-CTTATTATTCCATGCTGCAG-3′

650

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 2 μl DNA)

95 °C 5 min, 34× (95 °C 30 s, 53 °C 30 s, 72 °C 90 s), 72 °C 5 min

[11, 55]

Theileria spp.

Babesia spp.

Nested PCR 1

18S rDNA

5′-GTGAAACTGCGAATGGCTCATTAC-3′

5-AAGTGATAAGGTTCACAAAACTTCCC-3

1609–1523

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 1 μl DNA)

94 °C 1 min, 30× (98 °C 10 s, 55 °C 15 s, 68 °C 45 s), 68 °C 5 min

[10]

Nested PCR 2

5′-GGCTCATTACAACAGTTATAGTTTATTTG-3′

5′-CGGTCCGAATAATTCACCGGAT-3′

1544–1454

Anaplasma spp.

Nested PCR 1

Msp4

5′-ATGAATTACAGAGAATTGCTTGTAGG-3′

5-TTAATTGAAAGCAAATCTTGCTCCTATG-3′

849

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 5 μl DNA)

94 °C 4 min, 40× (94 °C 30 s, 55 °C 30 s, 72 °C 30 s), 72 °C 10 min

[56]

Nested PCR 2

5′-CTATTGGYGGNGCYAGAGT-3′

5-GTTCATCGAAAATTCCGTGGTA-3′

381

94 °C 4 min, 40× (94 °C 30 s, 56 °C 30 s, 72 °C 30 s), 72 °C 10 min

Borrelia spp.

Nested PCR 1

OspA

5′-CTTGAAGTTTTCAAAGAAGAT-3′

5′-CAACTGCTGACCCCTCTAAT-3′

487

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 2 μl DNA)

95 °C 5 min, 40× (95 °C 15 s, 55 °C 30 s, 72 °C 45 s), 72 °C 5 min

[57]

Nested PCR 2

5′-ACAAGAGCAGACGGAACCAG-3′

5′-TTGGTGCCATTTGAGTCGTA-3′

350

95 °C 5 min, 40× (95 °C 15 s, 58 °C 30 s, 72 °C 45 s), 72 °C 5 min

Bartonella spp.

Nested PCR 1

16S-23S rDNA ITS

5′-AAGTCGTAACAAGGT-3′

5′-TACTGGTKCGCTATCGGTCA-3′

1800

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 5 μl DNA)

95 °C 5 min, 40× (94 °C 30 s, 40 °C 45 s, 72 °C 4 min), 72 °C 10 min

[58]

Nested PCR 2

5′-TTTCTCTTTCTTCAGATGATG-3

′5′-AAAGCAGGTGCTCTCCCAGAAAGCAGGTGCTCTCCCAG-3

587

25 µl reaction volume (12.5 μl PCR master mix, 1 μl primers, 2 μl DNA)

95 °C 5 min, 30× (94 °C 30 s, 60 °C 30 s, 72 °C 40 s), 72 °C 5 min