Nucleic acid extraction from arthropods and selected livestock
blood samples
Arthropod pools were homogenised before nucleic acid extraction. Each
pool was placed in a 1.5-ml Eppendorf tube with pre-weighed scoops of
750 mg of 2.0-mm and 150 mg of 0.1-mm yttria stabilized zirconium oxide
(zirconia/yttria) beads (Biospec, USA), in which they were mechanically
disrupted using a Mini-Beadbeater-16 (BioSpec, Bartlesville, OK) for
60-90 seconds. Phosphate buffered saline (PBS) (360 µl) was added to
each tube, vortexed, and 210 µl of the resulting homogenate was
transferred to a 96-well specimen processing cartridge. DNA and RNA were
extracted using a MagNA 96 DNA and Viral NA Small Volume Kit (Roche
Applied Science, Penzberg, Germany) in a MagNA Pure 96 robot (Roche
Molecular Systems, California, USA). A sindbis virus culture isolate was
included as a positive extraction control and PBS was used as a negative
extraction control in each run. Total nucleic acid was eluted in 50 µl
of RNAse-free water.
Animal blood samples associated with arthropod pools identified as
positive for R. africae and CCHF virus were selected for pathogen
screening. Nucleic acids from blood samples were extracted using the
magnetic bead-based High Prep Viral DNA/RNA kit (MagBio Genomics,
Gaithersburg, USA). First, 200 µl of blood was added to 1.5-µl Eppendorf
tubes containing 528 µl of a lysis master-mix consisting of VDR lysis
buffer, isopropanol, and carrier RNA, and vortexed. Then 10 µl of
proteinase K and 10 µl of MAG-S1 magnetic beads were added and mixed
into solution by inversion. The subsequent steps were performed
according to the manufacturer’s instructions.