Application of PCR for rapid detection and serotyping of Salmonella spp. from porcine carcass swabs following enrichment in semi-solid agar

摘要

A new procedure for the detection and serotyping of Salmonella spp. based on PCR was developed and applied to porcine carcass swabs. Detection was based on a two-step enrichment using Buffered Peptone Water (BPW) and Modified Semi-solid Rappaport Vassiliadis (MSRV) agar followed by real time PCR targeting the genus specific hilA gene. In addition, pUC19 plasmid DNA was included as an extraction and internal amplification control (IAC). Serotyping consisted of multiplex PCR and capillary electrophoresis (CE) based on the method of Leader, Frye, Hu, Fedorka-Cray, & Boyle, 2009. To validate this method, 271 porcine carcass swabs were examined in tandem by the conventional culture method using MSRV (ISO 6579:2002; Annex D) followed by serotyping using slide agglutination and classification according to the White-Kauffmann-Le Minor scheme. Results showed 100% agreement between the conventional culture method and PCR, with 27 samples positive for Salmonella spp. by both methods. Using slide agglutination, full antigenic formulas were obtained for 25 of the 27 isolates (14 S. Typhimurium. 6 S. Infantis. 3 S. Derby, 1 S. Virchow and 1 S. Livingstone), while two isolates were not fully typeable and were designated S. Unnamed. PCR serotyping discriminated 7 different molecular code patterns, of which 2 were identical to those described by Leader et al. (2009), namely S. Typhimurium (ST) and S. Virchow (SV), found in 16 and one, respectively, of the 27 isolates. Both S. Unnamed isolates were identified as ST by the PCR method, therefore improving the identification of ST over the conventional method. The remaining 10 isolates shared four PCR patterns and although consistent codes were generally observed, discrepancies were observed compared to Leader et al. (2009). MSRV-PCR followed by serotyping using multiplex PCR and CE is rapid with final results obtained in 2 days from receipt of samples compared with 6-9 days using the conventional methods. The inclusion of the pUC19 plasmid DNA as an extraction and IAC demonstrated the robustness of the PCR procedure, with consistent Ct values obtained for both the hilA and pUC19 targets. This procedure could be particularly useful for surveillance and outbreak investigations by tracking sources of S. Typhimurium contamination, thus improving Salmonella control activities.