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DNA extraction protocol for rapid PCR detection of pathogenic bacteria
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.
Jeffrey D. Brewster
George C. Paoli
Escherichia coli O157
animal pathogenic bacteria
polymerase chain reaction
Analytical Biochemistry 2013 7 16 v.442 no.1
Journal Articles, USDA Authors, Peer-Reviewed
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