P1-23 Evaluation of a Real-time PCR Method to Detect Salmonella Enteritidis in Whole Shell Eggs

Monday, July 23, 2012
Exhibit Hall (Rhode Island Convention Center)
Robert Tebbs, Life Technologies, Austin, TX
Peyman Fatemi, Life Technologies, Austin, TX
Olga Petrauskene, Life Technologies, Foster City, CA
Arlene Nunez, Life Technologies, Foster City, CA
Craig Cummings, Life Technologies, Foster City, CA
Erin Crowley, Q Laboratories, Inc., Cincinnati, OH
Patrick Bird, Q Laboratories, Inc., Cincinnati, OH
Kiel Fisher, Q Laboratories, Inc., Cincinnati, OH
James Agin, Q Laboratories, Inc., Cincinnati, OH
Pius Brzoska, Life Technologies, Foster City, CA
David Goins, Q Laboratories, Inc., Cincinnati, OH
Manohar Furtado, Life Technologies, Foster City, CA
Catherine O'Connell, Life Technologies, Austin, TX
Introduction: Beginning in July of 2010, the U.S. Food and Drug Administration mandated routine environmental testing of poultry houses for presence of Salmonella Enteritidis. If SE is detected in the environment, then eggs must be tested prior to their distribution for sale.

Purpose: A new and rapid real-time PCR method was developed and evaluated against the FDA BAM culture method for detection of Salmonella Enteritidis in whole shell eggs.

Methods: Approximately 250 eggs were combined into a bulk sample for the uninoculated control, and a bulk lot consisting of approximately 900 eggs were inoculated with SE (ATCC 13706) at a concentration of 0.2-2 CFU per 1,000 g. Egg pools consisting of 20 eggs (1,000 g) were prepared. Twenty inoculated and 5 uninoculated egg pools were enriched according to FDA BAM.  A second set of 20 inoculated and 5 uninoculated egg pools were supplemented with 100 ml of 10X TSB per pool, and then incubated at 35°C for 24 hours. Real-time PCR was performed on the 7500 Fast using standard conditions (95°C for 10 min; 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds).

Results: Methods comparison showed that the real-time PCR method was equivalent to the FDA BAM method. For two independent studies, chi-square was 0 and 0.41. In addition, the two methods were compared on samples enriched according to the FDA BAM method on day 5 following overnight enrichment in TSB plus ferrous sulfate. Chi-square analysis indicated no difference between the two methods (χ2= 0 on two independent studies). No false-positive or false-negative results were observed using either enrichment method. Sample preparation was designed to be completely automated to give results in approximately 3 hours post enrichment.

Significance: The assay evaluated here was shown to be equivalent in performance to the FDA BAM culture procedure for the detection of Salmonella in eggs. Real-time PCR detection of SE in whole egg pools is simple, rapid, and more cost effective than the standard FDA BAM culture method.