PCR detection of PEDV
The samples were diluted with phosphate-buffered saline and centrifuged at 8,000×g for 10 min at 4°C and the supernatants were transferred into a 1.5 mL RNase-free tube. Viral RNA was extracted using the TIANamp Virus DNA/RNA Kit (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was carried out using PrimeScript™ IV 1st strand cDNA Synthesis Mix (TaKaRa). The full-length S gene of PEDV was amplified using EmeraldAmp® PCR Master Mix (TaKaRa). The S1 was amplified by two pairs of primers (S1-1U, 5’- ATCGTCAGAGGCATTTTTAA-3’; S1-1L, 5’-ATCCATCACCATTAAACGAA-3’; S1-2U, 5’-ATGTTGTGTTAGGCTTGTTG-3’; S1-2L, 5’- CACTAACAGGCGTGTTGTAA-3’), and the S2 was amplified by three primers (S2-U, 5’- CTGATTCTGGACAGTTGTTA-3’; S2-1L, 5’- TTGGACAGCATCCAAAGACA-3’; S2-2L, 5’- CTTCGAGACATCTTTGACAA-3’) (J. Chen et al., 2013). PCR products were purified and recovered using AxyPrep™ DNA Gel Extraction Kit (Axygen), then subjected to sequencing by Sangon Biotech company (Shanghai, China).