PEDV RNA Quantification from Samples

RNA from 200 µL of culture supernatant or fecal swab was isolated with the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and eluted in 50 µL. PEDV RNA was quantified using a Light Cycler 96 system (Roche, Indianapolis, IN, USA) with SensiFAST Probe No-ROX One-Step Kit (Bioline, Camarillo, TN, USA) using 20 µL of isolated RNA, 2× buffer, 10 µM forward primer (5′-CGCAAAGACTGAACCCA CTAATTT-3′), 10 µM reverse primer (5′-TTGCCTCTGTTGTTACTTGGAGAT-3′), and a 10 µM probe (5′-FAM-TGTTGCCATTGCCACGACTCCTGC-BHQ1-3′) [45 (link)]. The amplification steps were as follows: 10 min at 45 °C, 2 min at 95 °C, and 45 cycles of 15 s at 95 °C and 20 s at 60 °C.