Ambion magmax

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’: $\mathrm{Adjusted}\mathrm{C}\mathrm{q}=\left(35–\mathrm{sample}\mathrm{C}\mathrm{q}\right)$

In brief, viral RNA was extracted from 100 μl of fecal swab samples and eluted into 90 μl of elution buffer using the Ambion^® MagMAX^™ viral RNA isolation kit (Life Technologies) and a KingFisher^® 96 magnetic particle processor (Thermo-Fisher Scientific) following the procedures provided by the manufacturers.