Development of a Single-tube Multiplex Screening Assay for the Identification of Salmonella Heidelberg, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella spp. in the Poultry Environment

Introduction: Salmonella serovars Heidelberg, Enteritidis, and Typhimurium (HET) have been identified as increasingly significant human outbreak pathogens originating from poultry. To meet the need for efficient testing of poultry environmental samples, BioControl Systems developed a multiplex real time PCR-based screening method, Assurance GDS Salmonella HET Tq, to simultaneously identify each of the three HET serovars, as well as generic Salmonella spp. in a single reaction tube. A rapid immuno-magnetic separation step (IMS) is incorporated in the assay.

Purpose: To evaluate inclusivity and exclusivity of the method and the ability to predict semi-quantitative levels of contamination of environmental samples. 

Methods:  Inclusivity of the Assurance GDS Salmonella HET Tq assay was evaluated with 12 isolates of Salmonella Heidelberg, 18 Salmonella Typhimurium, and 14 Salmonella Enteritidis. Additionally, 17 isolates of non-HET Salmonella were analyzed to test exclusivity of the HET channels, and simultaneously the inclusivity of the Salmonella spp. detection. Thirty non-Salmonella isolates were used to test exclusivity of the HET assay.  Test strains were grown in non-selective broth, and diluted 10^-4 or 10^-2, respectively, for inclusivity and exclusivity. For semi-quantitative testing, cultures of Salmonella Heidelberg, Salmonella Enteritidis, and Salmonella Typhimurium were grown in TT medium and serially diluted. A standard curve was generated by plotting cycle threshold (Ct) vs. CFU/ml.

Results: Inclusivity testing of 61 Salmonella spp. resulted in the correct determination of each culture.  All 30 exclusivity strains were not detected.  Semi-quantitative standard curves were linear from 10⁴ to 10⁸ CFU /ml (R² >0.95, n=60). 

Significance: This single-tube multiplex screening method provides the poultry industry with a robust, efficient method to simultaneously identify three significant poultry-associated Salmonella serovars and Salmonella spp. in the poultry processing environment. The semi-quantitative characteristic of the assay may help assess contamination levels in poultry environmental samples.