Rapid detection of Salmonella in poultry environmental samples using real-time PCR coupled with immunomagnetic separation and whole genome amplification

Abstract

We evaluated the combination of immunomagnetic separation (IMS), multiple displacement amplification (MDA), and real-time PCR to detect Salmonella from poultry environmental samples. The limits of detection (LODs) of IMS-MDA real-time PCR with different culture enrichment hours (0, 4, 6, and 8 h) were determined in artificially inoculated litter samples from a specific pathogen-free (SPF) poultry farm. In addition, Salmonella detection rate of IMS-MDA real-time PCR with 8-h culture enrichment was compared with that of conventional real-time PCR and culture-based detection by analyzing 174 poultry environmental samples (boot swabs, drag swabs, and litter), and the levels of Salmonella in the samples were quantified using the most probably number method. The LODs of IMS-MDA real-time PCR with 0, 4 to 6, and 8-h enrichment were 10, 1, and 0.1 CFU/g, respectively. Salmonella was detected in 25 of the 174 environmental samples (14.4%) by IMS-MDA real-time PCR, compared with 24 (13.8%) by conventional real-time PCR and 19 (10.9%) by culturing. Cohen's kappa index indicated strong concordance (0.79) between IMS-MDA real-time PCR and culture detection. We demonstrated the potential of the IMS-MDA real-time PCR assay as a faster and more sensitive alternative to culture-based Salmonella detection from poultry environmental samples.

Keywords: Salmonella; IMS; MDA; detection; environmental sample; real-time PCR; whole genome amplification.

Figure 1

Flow diagrams of IMS-MDA real-time PCR, conventional real-time PCR, and culture method for detecting Salmonella in farm environmental samples in this study. “RVS”: Rappaport-Vassiliadis soya broth. “XLT4”: Xylose lysine Tergitol 4 agar.