Detection of pks+ Bacterial DNA

Conventional qualitative PCR was performed to evaluate the isolates of the fecal samples retrieved in the positive fraction of the magnetic separation. To detect pks+ bacterial DNA, primers amplifying a region of the clbB gene were used following the previously published protocols and primers (forward primer 5′-GATTTGGATACTGGCGATAACCG-3′ and reverse primer 5′-CCATTTCCCGTTTGAGCACAC-3′) (2 (link), 47 (link)). About 20 mL of reaction mixture for conventional qualitative PCR consisted of 1 µL of DNA (DNA concentrations are summarized in Table S5), each 0.6 µL of 10 µmol/L forward and reverse primer solutions, deoxynucleotide mixture, the DNA polymerase Master Mix RED (Ampliqon), and H₂O. The PCR conditions were 10 min at 95°C, 30 cycles of 45 s at 95°C, 45 s at 54°C, and 1 min at 72°C, and a final step of 7 min at 72°C. E. coli Nissle 1917 was selected as the positive control for pks+ detection and E. coli LMG2092 as the negative control.