Amplification of tick-borne pathogen DNA

Individual ticks were mechanically homogenized by crushing with liquid nitrogen using disposable micro pestle and the DNA was extracted using the Tissue and Bacterial DNA Purification Kit (EURx DNA, Gdansk, Poland) according to the manufacturer’s protocols. All Polymerase Chain Reaction (PCR) amplifications were conducted with final volumes of 25 μl with 2.5 μl of template DNA, while negative and positive controls for each pathogen were used. With the exception of Francisella tularensis and protozoa, ticks were molecularly screened for pathogens by real-time-PCR using Evagreen master mix (Biotium, State, USA), while suspected samples were subjected to PCR. For the detection of F. tularensis and Leishmania a real-time-PCR taqman probe was used. For the identification of Babesia, the conventional PCR was used. All positive samples were sequenced. The primers BJ1 and BN2 amplifying Babesia spp., detected also Theileria spp., Hepatozoon spp. and H. mauritanica. The PCR methods, target genes and primer sequences used can be seen in Table 1 [31–41].