A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Using Real-time PCR |
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Authors: | Trina Chua Arvind A Bhagwat |
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Affiliation: | (1) Produce Quality and Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, 10300 Baltimore Avenue, Building 002, Room 117, BARC-W, Beltsville, MD 20705-2350, USA;(2) Present address: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, Singapore, 599489, Singapore |
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Abstract: | DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples,
DNA isolation from food samples, particularly from fresh and fresh-cut produce has remained a formidable task and has hampered
the sensitivity and accuracy of molecular methods. We utilized a commercially available filter-based DNA isolation method
(FTA) in conjunction with real-time PCR-based detection of Salmonella spp. and Listeria monocytogenes. The protocol uses filter paper discs impregnated with a patented chemical formulation that lyses cells, immobilizes DNA,
and protects it from degradation. Use of the FTA method in conjunction with real-time PCR for the detection of Salmonella spp. and L. monocytogenes was compared with two commercially available DNA isolation procedures that are commonly used for high throughput real-time
PCR pathogen detection systems. Both pathogens were successfully detected from artificially inoculated fresh and fresh-cut
produce such as alfalfa sprouts, cilantro, green onion, broccoli, prepacked mixed salad, and spinach at low cell numbers (four
to seven colony forming units per 25 g initial inoculum level before enrichment). The FTA protocol had distinct advantages
of simplicity, biosafety, and compatibility for high throughput screening. This DNA preparation protocol was rapid, sensitive,
required minimal handling, and reduced interference from produce-associated inhibitors of real-time PCR.
Mention of brand names does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar
nature not mentioned. |
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Keywords: | Pathogen Detection Food-borne Food Safety Fresh Produce Food Microbiology |
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