45 ticks (n = 9) significantly differed for B. burgdorferi (s.l.), Rickettsia spp., and "Ca. N. mikurensis", but were not related to the habitat type. Three hundred fifty eight out of 1078 I. ricinus ticks (33.2%) tested positive for at least one pathogen. Thereof, about 20% (71/358) were carrying two or three different potentially disease-causing agents. Using next generation sequencing, we could detect true pathogens, tick symbionts and organisms of environmental or human origin in ten selected samples. Conclusions Our data document the presence of pathogens in the (sub-) urban I. ricinus tick population in Switzerland, with carrier rates as high as those in rural regions. Carriage of multiple pathogens was repeatedly observed, demonstrating the risk of acquiring multiple infections as a consequence of a tick bite."/>
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Table 2 Primer sequences and annealing temperatures of Sanger sequencing reactions

From: Prevalence of tick-borne pathogens in questing Ixodes ricinus ticks in urban and suburban areas of Switzerland

Species Primer sequences (5'–3') Amplicon length (bp) Amplicon position (reference sequence) Annealing T (°C) Reference
Fist-step PCR     
Borrelia spp. locus: 5S-23S intergenic spacer (forward: GAG TTC GCG GGA GAG TAG GTT ATT; reverse: TCA GGG TAC TTA GAT GGT TCA CTT CC 420 3063–3483 (JX564636.1) 64 [111]
Babesia spp. locus: 18S ribosomal RNA gene (forward: GTC TTG TAA TTG GAA TGA TGG; reverse: TAG TTT ATG GTT AGG ACT ACG) 489 466–955 (AJ439713) 58 [112]
Rickettsia spp. locus: 23S–5S intergenic spacer (forward: GAT AGG TCR GRT GTG GAA GCA C; reverse: TCG GGA YGG GAT CGT GTG TTT C) 388 1–388 (AY125012) 68 [113]
 Ticks locus: cytochrome c oxidase (forward: ACW AAY CAY AAA GAC ATT GGA AC; reverse: WGG ATG CCC RAA RAA TCA AAA T) 704 1242–1946 (KF197132) 48 [114]
Second-step PCR     
Borrelia spp. locus: 23S-5S intergenic spacer (forward: GGA GAG TAG GTT ATT GCC AG; reverse: GGT TCA CTT CCC CTG GTA TC) 396 3073–3468 (JX56436.1) 64
Babesia spp. locus: 18S ribosomal RNA gene (forward: GTA ATT GGA ATG ATG GTG AC; reverse: GTT AGG ACT ACG ACG GAA TC) 475 471–946 (AJ439713) 58
Rickettsia spp. locus: 23S-5S intergenic spacer (forward: CAG TAA TGT GTG TAG CTA AC; reverse: ATC GTG TGT TTC ACT CAT GC) 356 22–378 (AY125012) 58
 Ticks locus: cytochrome c oxidase (forward: AYC AYA AAG ACA TTG GAA CWA T; reverse: GCC CRA ARA ATC AAA ATA RAT G) 686 1245–1933 (KF197132) 48