Discrimination of the ΔaroA vaccine strain from other E. coli strains and non-E. coli strains using PCR.

The specificity of the primers targeting either the E. coli ΔaroA gene (A), the WT E. coli aroA gene (B), the previously published primers targeting both the WT and ΔaroA gene (total aroA) (D) or simultaneous detection of the ΔaroA and xanQ gene (C), was screened against a total of 132 E. coli strains and 7 non-Escherichia strains. (A) PCR specific for the ΔaroA gene. Amplification only occurred for the vaccine strain, while none of the commensal E. coli strains or non-Escherichia strains was amplified. The picture shows the results from primer pair aroA_3, which results in a band at 281bp when the aroA mutation is present. All other tested ΔaroA specific primers showed the same specificity. (B) The aroA primers targeting the WT gene (aroA_WT) result in a band at 660 bp in commensal E. coli, whereas no band occurred when the aroA mutation was present or in the non-Escherichia strains. (C) Duplex PCR with aroA_3 and xanQ_2 primers. The E. coli-specific xanQ primers result in a band at 97 bp, whereas the band at 281 bp indicates ΔaroA. Only for the ΔaroA E. coli strain, the two bands are present. (D) Primer pair aroA_LR (La Ragione et al., 2013, Table 1) is not specific for the ΔaroA mutation and results in an amplicon of 1236 bp for wild type E. coli, or 1161 bp for the ΔaroA vaccine strain. The pictures show a representative selection of the total number of strains used to assess the primer specificity. Samples: 1: blank control 2: E. coli O78:K80 ΔaroA (vaccine strain), 3: E coli O78:K80 WT, 4: E. coli Nissle 1917, 5: APEC_1 (chicken intestine), 6: APEC_2 (turkey intestine), 7: APEC019 (chicken intestine), 8: APEC023 (chicken intestine), 9: 220115–1 (chicken intestine, aromatic amino acid auxotrophic E. coli), 10: 220115–10 (chicken intestine, aromatic amino acid auxotrophic E. coli), 11: 06114–21 (chicken intestine, aromatic amino acid auxotrophic E. coli), 12: 7-35s (chicken intestine, aromatic amino acid auxotrophic E. coli), 13: 3-44s (chicken intestine, aromatic amino acid auxotrophic E. coli), 14: Salmonella Enteritidis, 15: Salmonella Typhimurium, 16: Clostridium perfringens NE18, 17: Clostridium perfringens CP20, 18: Bacillus subtilis DSM 29784. The complete list of tested strains can be found in S1 Table. Electrophoresis was performed on 1.5% agarose gel. GeneRulerTM 100 bp Plus DNA Ladder was used as a molecular weight marker.