Rapid Detection of Salmonella in Raw Chicken Breast Using Real-time PCR Combined with Immunomagnetic Separation and Whole Genome Amplification

Introduction: Nontyphoidal Salmonella enterica is one of the leading causes of foodborne illness worldwide, accounting for more than one million cases in the U.S each year. A number of real-time PCR-based detection for Salmonella have been developed; due to the low abundance and uneven distribution of Salmonella in food, they are typically preceded by enrichment, which constitutes a bottleneck to enabling same-day analysis without compromising detection sensitivity.

Purpose: We developed a real-time PCR-based detection combined with targeted cell capture by immunomagnetic separation (IMS) and highly efficient amplification of genomic DNA by multiple displacement amplification (MDA) for Salmonella enterica Serotype Enteritidis (SE) from chicken breast.

Methods: The limits of detection (LOD) of IMS-MDA real-time PCR were determined in artificially SE-contaminated chicken breast (n=18) after 0, 2, and 4-hour enrichment. In addition, the equivalence of this method in detecting refrigeration-stressed SE on chicken breast (n=90) was assessed by comparing with conventional real-time PCR and culture-based approach.

Results: The LODs of IMS-MDA real-time PCR for detecting SE in chicken meat were 10 CFU/g, 1 CFU/g, and 0.1 CFU/g after 0, 2, and 4-hour enrichment, respectively. The detection rate of IMS-MDA real-time PCR appeared to be almost on par with that of culture-based detection. Salmonella was detected at medium (1 CFU/g) and high (10 CFU/g) inoculum levels in 27 and 29 out of 30 samples after 4-h enrichment, respectively, and showed no statistical difference (P > 0.05) at the low (0.1 CFU/g) inoculum level. In addition, IMS-MDA real-time PCR yielded significantly more positive results compared to conventional real-time PCR with statistical difference (P< 0.05).

Significance: We demonstrated the potential of IMS-MDA real-time PCR as a rapid, sensitive, and affordable method for detecting Salmonella in food. The successful application of this method suggested that this technique may be used for other pathogens and food samples.