Molecular identification of ticks, lice, and fleas
Molecular identification was performed on 15 single specimens for which
morphologic identification to species level was equivocal. We amplified
three target genes: the internal transcribed spacer-2 (ITS2), cytochrome
oxidase 1 (CO1), and 16S ribosomal (r)RNA (Supplementary Table 1). The
PCRs were performed in a SimpliAmp PCR Thermal Cycler (Applied
Biosystems, Singapore) in 10-µl reactions that consisted of 2 µl of 5x
HOT FIREPol® Blend Master Mix (Solis BioDyne, Estonia), 2 µl of
template, and 0.5 µl of 10 µM primer. Molecular grade water was included
as a negative control on each run. The cycling conditions have been
described before in detail (Mwamuye et al., 2017), with the exception
that the final extension step for the three fragments was seven minutes.
Amplicons of the correct size were visualised alongside Quick-Load®
100-bp DNA Ladder (Biolabs, UK) by electrophoresis on 1.6 % ethidium
stained agarose gels under UV light. Bi-directional sequencing of
amplicons purified by Exo 1-rSAP combination (Biolabs, UK) was performed
by Macrogen (Netherlands). Sequence chromatograms were inspected,
edited, and aligned using Geneious Prime version 2019.0.4 software
(Biomatters, New Zealand). The resulting sequence contigs were used in
nucleotide BLAST searches (Altschup et al., 1990) against the GenBank nr
database (www.ncbi. nlm.nih.gov/blast) to identify sequence matches.