Simultaneous detection of Salmonella spp and Escherichia coli O157:H7 by multiplex PCR |
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Authors: | P M Fratamico T P Strobaugh |
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Institution: | (1) Microbial Food Safety Research Unit, US Department of Agriculture, Eastern Regional Research Center, Agricultural Research Service, 600 E Mermaid Lane, Wyndmoor, PA 19038, USA, US |
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Abstract: | Contamination of foods with pathogens such as Escherichia coli O157:H7 and Salmonella is a major concern worldwide and rapid, sensitive, and reliable methods are needed for detection of these organisms. Since
these pathogens can contaminate similar foods and other types of samples, a multiplex polymerase chain reduction (PCR) was
designed to allow simultaneous detection of both E. coli O157:H7 and Salmonella spp directly from enrichment cultures. Samples of apple cider, beef carcass wash water, ground beef, and bovine feces were
inoculated with both E. coli O157:H7 and S. typhimurium at various bacterial levels. Following enrichment culturing for 20–24 h at 37°C in modified EC broth or buffered peptone
water both containing novobiocin, the samples were subjected to a DNA extraction technique or to immunomagnetic separation
then tested by the multiplex PCR assay. Four pairs of primers were employed in the PCR: primers for amplification of E. coli O157:H7 eaeA, stx
1/2 and plasmid sequences and for amplification of a portion of the Salmonella invA gene. Four fragments of the expected sizes were amplified in a single reaction and visualized following agarose gel electrophoresis
in all the samples inoculated with ≤ 1 CFU g−1 or ml−1. Results can be obtained in approximately 30 h. The multiplex PCR is a potentially powerful technique for rapid and sensitive
co-detection of both pathogens in foods and other types of samples.
Received 28 December 1997/ Accepted in revised form 19 March 1998 |
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Keywords: | : immunomagnetic separation bovine feces carcass wash water apple cider ground beef |
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