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P2-60 Evaluation of DNA Extraction and Real-time PCR Screening Method for Listeria monocytogenes and Listeria spp. from Cantaloupe Peel and Queso Fresco Cheese

Tuesday, August 2, 2016
America's Center - St. Louis
Ken Yoshitomi, U.S. Food and Drug Administration, Office of Regulatory Affairs, Office of Regulatory Science, Rockville, MD
Karen Jinneman, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
Kun Liu, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
Patricia Nguyen, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
Khamphet Nabe, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
June Wetherington, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
Doan Nguyen, U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Northwest, Bothell, WA
Introduction: Listeria monocytogenes is a significant foodborne pathogen.  Detection traditionally includes enrichment with isolation on selective media. Real-time PCR (qPCR) screening would assist rapid detection in food samples. Optimal DNA template preparation removes PCR inhibitors, concentrates target and improves overall qPCR sensitivity.

Purpose: Evaluate semi-automated DNA extraction with qPCR detection for L. monocytogenes and Listeria species from cantaloupe peel and soft cheese.

Methods: Two runs per matrix included 20 low (≈0.5 CFU/g), 5 high ( ≈5 CFU/g)  L. monocytogenes inoculum levels and 5 uninoculated sample replicates. Surface inoculated cantaloupes were air dried and aged 48 h. Cheese samples were inoculated and aged 72 h at 4°C.  Samples analyzed per BAM. Also, 48-h enrichments screened by preparing DNA template on MagMax Express 96 and analyzed by LIS-7500 multiplex qPCR assay on AB 7500 platform.  Three replicate qPCR tests were run from each DNA template. The LIS-7500 assay targets iap gene in L. monocytogenes and Listeria spp. plus an internal amplification control.

Results: The qPCR screen detected Listeria in 15/20 and 14/20 at low, 4/5 and 5/5 at high inoculum levels and 0/5 and 0/5 for the uninoculated samples for cantaloupe peels runs 1 and 2.  Similarly, L. monocytogenes was culturally recovered from 15/20 and 13/20 at the low and 4/5 and 5/5 at the high inoculum and 0/5 and 0/5 for uninoculated runs 1 and 2, respectively. The first cheese run had equivalent results for positive qPCR screen and cultural recovery with 20/20 at low, 5/5 at high and 0/5 for uninoculated samples.  In the second run 13/20 at low, 5/5 at high and 0/5 uninoculated samples had L. monocytogenes detected by qPCR.  Culturally, L .monocytogenes was recovered from 19/20, 5/5 and 0/5 at low, high and uninoculated levels, respectively.    

Significance: Rapid Listeria species and specifically L. monocytogenes detection from contaminated food could streamline and increase sample throughput.

Back to: Poster Session 2 – Low-water Activity, Laboratory and Detection Methods, Epidemiology, Produce, Pre-harvest, Dairy and Beverages, Food Defense, General Microbiology
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