Evaluation of a real-time PCR assay for rapid detection of Listeria monocytogenes in artificially contaminated soft cheese and environmental surface samples
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Contributing OfficeCenter for Food Safety and Applied Nutrition
Abstract
Introduction
Listeria monocytogenes is an opportunistic foodborne pathogen that can be associated with a variety of food and environmental sources. Real-time PCR allows rapid and accurate screening of this foodborne pathogen.
Purpose
A real-time PCR method, originally developed to confirm culture identity and later modified to include an internal amplification control and to use on the Applied Biosystems 7500 FAST instrument, was evaluated for rapid screening of L. monocytogenes from cheese and environmental samples and compared with the current Food and Drug Administration (FDA)’s Bacteriological Analytical Manual (BAM).
Methods
Soft cheese (queso fresco) and environmental surfaces were artificially inoculated with low levels of L. monocytogenes. After a 48 h of enrichment described in the FDA BAM, selective agar plates were used for qualitative detection. The 48 h-enriched test portions were also used for DNA extraction on a MagMax/Kingfisher apparatus that is designed to process 96 samples per run. Two manual DNA extraction methods were also evaluated. A real-time PCR that can simultaneously detect Listeria spp. and L. monocytogenes was performed on the Applied Biosystems 7500 FAST instrument.
Results
Real-time PCR screening and BAM cultural scheme generated 100% consistent results in 53 environmental test portions that contained ca. 1 to 10 CFU per test portion, and 15 soft cheese test portions that contained ca. 1 to 5 CFU per test portion. The uninoculated controls were negative by either real-time PCR or cultural analysis. Manual DNA extraction could be an alternative to the automatic DNA extraction.
Significance
This detection method based on BAM enrichment and real-time PCR provides highly sensitive screening for L. monocytogenes in artificially contaminated soft cheese and environmental samples. Evaluation and further validation of this method should help to establish an improved L. monocytogenes surveillance strategy with considerably reduced time and costs.