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).