Smart cycler detection system

Reagents for the PEDV qRT-PCR assay were obtained from a commercially available reaction kit (QIAGEN One Step RT-PCR reagent kit; QIAGEN, Valencia, CA, U.S.), with the addition of magnesium (Sigma-Aldrich Inc., St. Louis, MO, U.S.). The PEDV primers and probes were developed according to a previously published method [33 (link)] and ordered from a commercial supplier (IDT, Iowa City, IA, U.S.). The RT-PCR reaction mix consisted of 5 μL 5× Reaction Buffer, 1 μL nucleotide triphosphates, 2 μL of 25 μM/mL MgCl₂, 5 μL nuclease-free water, 2 μL PEDV forward and reverse primers (10 μM each), 1 μL PEDV HEX-labeled probe (5 μM), 1 μL One Step RT-PCR Enzyme mix, and 8 μL extracted RNA. Each RT-PCR sample was analyzed on a Cepheid Smart Cycler Detection System (Cepheid, Sunnyvale, CA, U.S.) under the following conditions: 50 °C for 30 min; 95 °C for 15 min; and 45 cycles of 94 °C for 30 s, 60 °C for 60 s with optics on, and 72 °C for 30 s. Validated PCR positive controls consisting of PEDV RNA and negative extraction controls were included in each run. Samples were considered positive if the mean fluorescence exceeded 30 fluorescent units prior to 40 cycles and negative and positive PCR controls were properly classified.