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P1-45 Development and Preliminary Evaluation of a Real-time PCR Assay for the Detection of Listeria monocytogenes

Monday, August 4, 2014
Exhibit Hall D (Indiana Convention Center)
Seth Blumerman, DuPont Nutrition & Health, Wilmington, DE
Lois Fleck, DuPont Nutrition & Health, Wilmington, DE
Daniel DeMarco, DuPont Nutrition & Health, Wilmington, DE
Stephen Varkey, DuPont Nutrition & Health, Wilmington, DE
Mark Jensen, DuPont Nutrition & Health, Wilmington, DE
Andrew Farnum, DuPont Nutrition & Health, Wilmington, DE
Teresa Brodeur, DuPont Nutrition & Health, Wilmington, DE
Timothy Dambaugh, DuPont Nutrition & Health, Wilmington, DE
Gongbo Wang, DuPont Nutrition & Health, Shanghai, China
George Tice, DuPont Nutrition & Health, Wilmington, DE
Introduction:   Listeria monocytogenes is a significant foodborne pathogen.  Because outbreaks of listeriosis in humans predominantly stem from the ingestion of contaminated food, there is a need for sensitive, accurate, and rapid test methods for the detection of L. monocytogenes in a wide range of food and environmental samples.

Purpose: The purpose of this study was to evaluate a newly developed real-time PCR assay capable of detecting all serotypes of Listeria monocytogenes.  Inclusivity and exclusivity testing, along with assay sensitivity, were assessed in these studies.

Methods:  Cellular lysates from overnight cultures were used in these studies, with lysis carried out using a modified protocol.  Cell lysates from overnight BHI cultures were used to test inclusivity at a level approximately 1-log above the limit of detection of the assay (cultures diluted to approximately 104 - 105 CFU/ml), and exclusivity (cultures at ≥ 108 - 109 CFU/ml).  The sensitivity of the real-time assay was tested using cell lysates from serially diluted and enumerated cell cultures (diluted to approximately 10 CFU/ml) inoculated into a variety of food matrices. 

Results: The sensitivity of the assay was shown to be ≤ 1x104 CFU/ml.  The assay was 100% inclusive for all the strains tested (n = 53).  Exclusivity was also determined to be 100% against other Listeriaspp. and closely related genera (n = 77).

Significance: These results demonstrate the feasibility of a new real-time PCR assay, with an alternative lysis method, for the detection of L. monocytogenes. This new assay allows for a more rapid time-to-result for the testing of food and environmental samples, while maintaining the simplicity, accuracy and reliability of the BAX® System method.

Back to: Poster Session - Applied Laboratory Methods, Produce, Low-water Activity, Beverages and Water
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