Kingfisher 96 magnetic particle processor

In brief, 90 μl of viral RNA was eluted from rectal swabs, fecal samples and oral fluid specimens using the Ambion® MagMAX™ viral RNA isolation kit (Life Technologies, Carlsbad CA USA) and a KingFisher® 96 magnetic particle processor (Thermo-Fisher Scientific) following the procedures provided by the manufacturers. Samples were tested for PEDV using a PEDV N gene-based rRT-PCR described in Madson et al. [7 (link)] and performed routinely at the Iowa State University-Veterinary Diagnostic Laboratory (ISU-VDL SOP 9.5263). The forward primer sequence was 5′-CGCAAAGACTGAACCCACTAACCT-3′, the reverse primer sequence was 5′-TTGCCTCTGTTGTTACTTGGAGAT-3′, and probe sequence was 5′-FAM-TGTTGCCAT/ZEN/TACCACGACTCCTGC-Iowa Black-3′. The eluted RNA, primers, and probe were mixed with commercial reagents TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) and the rRT-PCR reactions were conducted on an ABI 7500 Fast instrument (Life Technologies) in fast mode as follows: 1 cycle at 50 °C for 5 min, 1 cycle at 95 °C for 20 s, 40 cycles at 95 °C for 3 s, and 60 °C for 30 s. The results were analyzed using an automatic baseline setting with a threshold at 0.1. Quantification cycle (Cq) values < 35 were considered positive for the corresponding coronavirus. Data were reported as ‘adjusted Cqs’: $\mathrm{Adjusted}\mathrm{C}\mathrm{q}=\left(35–\mathrm{sample}\mathrm{C}\mathrm{q}\right)$

Two aliquots of each tissue sample collected at necropsy were thawed and individually macerated in tissue culture media, using a TissueLyser bead beater (Qiagen, Valencia, CA) and stainless steel beads (Qiagen cat. no. 69989). Total RNA was extracted from tissue macerates, serum, and OPF samples using Ambion’s MagMax-96 Viral RNA Isolation Kit (Ambion, Austin, TX) on a King Fisher-96 Magnetic Particle Processor (Thermo Scientific, Waltham, MA). Extracted RNA was analyzed using quantitative real-time RT-PCR (RT-qPCR), targeting the 3D region of the FMDV genome [40 (link)] with forward and reverse primers adapted from Rasmussen et al [41 (link)], and chemistry and cycling conditions as previously described [42 (link)]. Cycle threshold values were converted to FMDV RNA copies using an equation derived from analysis of serial 10-fold dilutions of in vitro synthesized FMDV RNA of known concentration. The equations of the curve of RNA copy numbers versus Ct values were further adjusted for the average mass of tissue samples and specific dilutions used during processing of samples.