DNA extraction protocol for rapid PCR detection of pathogenic bacteria |
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Authors: | Jeffrey D. Brewster George C. Paoli |
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Affiliation: | Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, Philadelphia, PA 19118, USA |
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Abstract: | Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT + Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. |
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Keywords: | DNA extraction PCR Bacteria Escherichia coli O157:H7 Listeria monocytogenes |
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