Molecular-phylogenetic analyses of Ixodes species from South Africa suggest an African origin of bird-associated exophilic ticks (subgenus Trichotoixodes)

Table 2 Oligonucleotide sequences and cycle parameters of taxonomic PCRs used in this study

Target gene (approx. length)	Primers, probes (5'-3')	initial denaturation	cycle denaturation	cycle annealing	cycle extension	final extension	Cycle n =	References
cox1 (710 bp)	LCO1490 (GGT CAA CAA ATC ATA AAG ATA TTG G) HCO2198 (TAA ACT TCA GGG TGA CCA AAA AAT CA)	95 °C, 5 m	94 °C, 40 s	48 °C, 1 m	72 °C, 1 m	72 °C, 10 m	40	[24]
16S rRNA gene (460 bp)	16S + 1 (CTG CTC AAT GAT TTT TTA AAT TGC TGT GG) 16S-1 (CCG GTC TGA ACT CAG ATC AAG T)	95 °C, 5 m	94 °C, 40 s	51 °C, 1 m	72 °C, 1 m	72 °C, 10 m	40	[25]
18S rRNA gene (1300 bp)	NS1 (GTA GTC ATA TGC TTG TCT C) NS4a (GCC CTT CCG TCA ATT CCT TTA AG)	95 °C, 5 m	94 °C, 40 s	52 °C, 1 m	72 °C, 1 m	72 °C, 10 m	40	[17, 26]
18S rRNA gene (780 bp)	18S-F (CAT TAA ATC AGT TAT GGT TCC) 18S-R (CGC CGC AAT ACG AAT GC)	95 °C, 5 m	94 °C, 30 s	52 °C, 30 s 50 °C, 30 s 48 °C, 30 s 46 °C, 30 s	68 °C, 1 m	68 °C, 5 m	5 5 5 25	[27]
28S rRNA gene (700 bp)	28ScF (GTG GTA GCC AAA TGC CTC GTC ATC) 28SR (GAA TTC TGC TTC ACA ATG ATA GGA AGA GCC)	95 °C, 5 m	94 °C, 40s	58 °C, 1 m	72 °C, 1m	72 °C, 10 m	40	[28, 29]
28S rRNA gene (330 bp)	Tick-28S-C2-F (GCG GCG AGT AGG TCG GTA ACC) Tick-d9-D3-R (ACG TCA GAA TCG CTT CGG A)	95 °C, 5 m	95 °C, 30 s	60 °C, 30 s	72 °C, 1m	72 °C, 5 m	40	[30]

Reaction components: volume 25 µl, containing 1 U (0.2 µl) HotStarTaq Plus DNA polymerase, 2.5 µl 10 × CoralLoad reaction buffer (including 15 mM MgCl₂), 0.5 µl PCR nucleotide Mix (0.2 mM each), 0.5 µl (1 µM final concentration) of each primer, 15.8 µl ddH₂O and 5 µl template DNA

ISSN: 1756-3305