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P1-91 Validation of a FDA-developed Multiplex Real-time Quantitative PCR (qPCR) for the Identification of Salmonella Enteritidis Using ABI 7500 Fast System

Monday, August 1, 2016
America's Center - St. Louis
Hua Wang, U.S. Food and Drug Administration-CFSAN, College Park, MD
Chorng-Ming Cheng, U.S. Food and Drug Administration, Irvine, CA
Anna Laasri, U.S. Food and Drug Administration-CFSAN, College Park, MD
Kai-Shun Chen, U.S. Food and Drug Administration, Irvine, CA
Andrew Jacobson, U.S. Food and Drug Administration, College Park, MD
Thomas Hammack, U.S. Food and Drug Administration, College Park, MD
Introduction: Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis) has become the primary cause of salmonellosis worldwide, accounting for 36% of the 403 outbreaks in 1998 to 2008 in United States. Contaminated eggs and poultry are major sources of human Salmonella infections; however, outbreaks have also been associated with beef, turkey, sprouts, nuts, leafy and vine stalk vegetables.

Purpose: To validate a FDA-developed multiplex real-time qPCR using ABI 7500 Fast system for identifying Salmonella Enteritidis isolates and detecting Salmonella Enteritidis in food and environmental samples.

Methods: Our real-time qPCR assay detects the presence of a 262-bp fragment of the Salmonella-specific invA gene and a 159-bp fragment of the Salmonella Enteritidis-specific SDF gene using custom-designed primers and TaqMan probes, along with a custom-designed internal control. We tested 105 Salmonella Enteritidis strains (15 phage types), 130 non-Enteritidis Salmonella strains (126 serotypes), and 30 non-Salmonella strains. Assays on pine nuts naturally contaminated with S. Enteritidis and chicken house drag swabs artificially contaminated with S. Enteritidis were performed on 24-h preenriched cultures.  

Results: The assay correctly identified all 105 Salmonella Enteritidis. Three strains of the 130 non-Enteritidis Salmonella tested positive for the SDF genes: one of two Salmonella Give strains, one of two Salmonella Typhimurium strains, and one of two Salmonella Nottingham strains. These anomalous strains have been sequenced for further analysis. All 30 non-Salmonella were negative for the SDF genes. Our 24-h qPCR results from pine nut and drag swab samples showed 100% agreement to the results of BAM culture analysis and molecular serotyping assays.

Significance: This validated real-time qPCR will provide FDA an effective tool for specially detecting Salmonella Enteritidis in high-throughput food and environmental samples.

Back to: Poster Session 1 – Produce, Meat, Poultry and Eggs, Non-microbial Food Safety; Laboratory and Detection Methods; Communication Outreach and Education; Seafood; Antimicrobials; Food Toxicology
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