A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Usin
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A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Using Real-time PCR Trina Chua & Arvind A. Bhagwat
Received: 31 January 2008 / Accepted: 14 April 2008 / Published online: 13 June 2008 # Springer Science + Business Media, LLC 2008
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
Mention of brand names does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned. T. Chua : A. A. Bhagwat (*) 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 e-mail: [email protected] Present address: T. Chua School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, Singapore 599489, Singapore
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. Keywords Pathogen Detection . Food-borne . Food Safety . Fresh Produce . Food Microbiology
Introduction Advances in molecular biology have led to the use of realtime PCR as an efficient and reproducible method for detecting pathogens (McKillip and Drake 2004; Rijpens and Herman 2002; Rodriguez-Lazaro et al. 2004a, 2007). Rather than relying on culture and biochemical properties, PCR-based assays offer more rapid, sensitive, and specific detection capabilities. Conventional and well-established technologies such as single- and double-step enrichment of human pathogens combined with immunomagnetic separation have given promising results (Jaykus 2003; Fluit et al. 1993; Shelton and Karns 2001; Josefsen e
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