Harris 3 mm micro puncher

DNA was extracted from Anopheles mosquitoes using the DNeasy 96 Blood & Tissue Kit (Qiagen) in accordance with the manufacturer's instructions for DNA purification from insects. Briefly, mosquitoes were placed in 1.5‐mL Eppendorf tubes containing 180 μL of Buffer ATL (Qiagen) and 20 μL of proteinase K (Qiagen) and crushed using a 1‐mL pipette tip or mortar. Samples were incubated at 56 °C overnight in an incubator. DNA was further extracted in accordance with the manufacturer's instructions. DNA was extracted from 1900 individual mosquitoes and the remainder (3500 mosquitoes) were pooled (five mosquitoes per pool) to reduce the cost of DNA extraction. After DNA extraction, 50 μL of the DNA extract was treated with a One Step™ PCR inhibitor removal kit (Zymo Research, Irvine, CA, U.S.A.) before PCR amplification.
DNA was extracted from CloneSaver FTA filter paper cards (GE Healthcare) using the Allprep DNA/RNA mini kit (Qiagen). Briefly, two or three disks of the dried blood spots were punched out using a Harris 3‐mm micro‐puncher (GE Healthcare). Two or three discs were mixed in a 1,5‐mL Eppendorf tube with 350 μL of RLT buffer (Qiagen) containing 1% β‐mercaptoethanol and incubated for 1 h at 37 °C with shaking (1000 rpm). Afterwards, DNA/RNA was extracted in accordance with the manufacturer's instructions.

Extraction of viral RNA from dried blood spots (DBS) stored on FTA cards were based on previously described methods [17 ]. A Harris 3 mm micro-puncher (GE Healthcare Life Sciences) was used to remove 1 to 3 punches of the DBS on FTA cards and the punches were placed into a 96-well plate with 70 μl of RNA rapid extraction solution (Ambion, Austin, TX). The puncher was cleaned with 70% ethanol between samples. The 96-well plate was covered with an adhesive plate sealer and placed on a plate rocker for 20 min. at room temperature. Viral RNA was isolated from 50 μl of RNA rapid extraction solution containing FTA card punches or directly from blood diluted in PBS (back titration samples) using the MagMAX-96 viral RNA isolation kit (Ambion) and the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA) according to vendor’s protocols. The RNA was eluted in 50 μl of nuclease-free water.