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.