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Combination of Immunomagnetic Separation with Real-Time PCR for Rapid Detection of Salmonella in Milk, Ground Beef, and Alfalfa Sprouts

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Abstract:

A rapid, specific, and sensitive method for detecting Salmonella spp. in pasteurized milk, ground beef, and alfalfa sprouts was developed. The method combined immunomagnetic separation with a real-time PCR assay based on the double-stranded DNA binding dye SYBR Green I. The primers used produced a product with a melting temperature of 87 ± 0.5°C during the PCR assay by amplifying a 284-bp sequence from the invasive gene (invA) of Salmonella. The method was successful in detecting 20 Salmonella strains, but the expected PCR product was not formed by any of 11 other bacterial strains. To test this combined method for the monitoring of Salmonella , Salmonella enterica serotype Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered peptone water, cells were removed by immunomagnetic separation and DNA extracted using the High Pure PCR template preparation kit. The DNA produced was used as a template in the real-time PCR assay. When spiked pasteurized milk, ground beef, and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of 1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13 h. These results indicate that the combination of immunomagnetic separation and realtime PCR assay was a highly specific and sensitive method for the rapid detection of Salmonella.

Document Type: Research Article

Publication date: March 1, 2005

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