Magna pure 96 dna and viral nucleic acid small volume kit

Genomic DNA was extracted from in vitro cell cultures and blood material using MPLC or MP96 instruments with their respective MagNa Pure LC DNA Isolation Kit – Large Volume or MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit (Roche Diagnostics, Mannheim, Germany), according to manufacturer’s instructions. Eluted DNA was tested for T. parva using the Hybrid II assay (Pienaar et al., 2011 (link)). Molecular biology work was done in a South African National Accreditation System (SANAS) accredited (V0017) and Department of Agriculture, Land Reform and Rural development (DALRRD) (DAFF-30) approved laboratory.

RNA was extracted on a MagNA Pure 96 extraction robot using the MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume kit (Roche, Basel, Switzerland) and the Pathogen 200 universal protocol version 4.0.

Mosquito trapping with CDC light and CO₂ traps was carried out annually (May through June) from 2012 to 2016 by the MDPH and collaborating mosquito control programs. Traps were set on average every 2 days, often in multiple localities, and traps were collected the following day. Mosquitoes were stored at 4°C, taxonomically sorted, and pooled (≤50 per pool). O. canadensis pools (N = 359) were homogenized in BA-1 medium, and total nucleic acid was extracted from 140 µl of homogenate using the MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume kit (Roche, Basel, Switzerland). An aliquot was confirmed negative for West Nile virus (WNV) and Eastern equine encephalitis virus (EEE) by RT–PCR [25 (link)]. As a positive control, RNA was extracted from a commercially-available JCV culture supernatant (ATCC^® VR-712™, lot #12507) using MagNA Pure Total Nucleic Acid I (Roche, Basel, Switzerland). To verify non-viability, extracted material was inoculated onto Vero cells grown at 37°C in BA-1 medium (Supplementary Data), subpassaged once and monitored for cytopathogenic effects for 14 days.