title: DNA extraction protocol for rapid PCR detection of pathogenic bacteria.
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.
authors.affiliation: Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, Philadelphia, PA 19118, USA. Electronic address: jeffrey.brewster@ars.usda.gov.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Brewster
authors.forename: George C
authors.initials: GC
authors.lastname: Paoli
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial
keywords: Bacteria; DNA extraction; Escherichia coli O157:H7; Listeria monocytogenes; PCR