2.1 Sample collection and DNA preparation
From 2016 to 2019, a total of 281 samples including 236 intestinal tissue samples and 45 fecal samples were collected from diarrheal piglets in 27 different pig farms in 18 cities of central China. Samples were diluted 1:10 with phosphate-buffered saline (PBS, pH7.4), and the suspensions were vortexed and centrifuged at 12 000 × g for 5 min at 4°C to collect the clarified supernatants. The viral DNA was extracted using an UNlQ-10 Column Virus Genomic DNA Isolation Kit (Sangon Biotech Shanghai Co, Ltd., China) according to the manufacturer’s instructions. The concentration of DNA was calculated and stored at -80°C.