The fecal swab and feces samples were vortexed for 2 min and centrifuged at 3000g for 5 min. For the intestinal samples, 1 g of the tissue was homogenized in 10 mL of phosphate buffer saline (PBS) and centrifuged at 3000g for 10 min. Viral RNA was extracted from 140 µL of the supernatants using the QIAamp Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions and eluted in 60 µL buffer AVE (Qiagen). PEDV RNA was detected using RT-PCR targeting the conserved regions of the PEDV nucleocapsid (N) protein gene as described by Kim et al. (2007) (link), with modifications to fit the ABI StepOne system. Briefly, using the TaqMan One-Step RT-PCR Kit (Applied Biosystems), RT-PCR was performed in a 25 µL reaction mix containing 4 µL RNA, 1 × RT-PCR enzyme mix, 1 × RT-PCR buffer, 40 pmol forward primer (5′-CGCAAAGACTGAACCCACTAA TTT-3′), 40 pmol reverse primer (5′-TTGCCTCTGTTGTTACTTGGAGAT-3′) and 12 pmol probe (Cy5-TGTT GCCATTGCCACGACTCCTGC-BHQ3). Amplification parameters were as follows: an initial step of 10 min at 50 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 20 s at 60 °C.