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DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Permanent URL:
http://handle.nal.usda.gov/10113/57845
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.
Author(s):
Jeffrey D. Brewster , George C. Paoli
Subject(s):
DNA , Escherichia coli O157 , Gram-negative bacteria , Gram-positive bacteria , Listeria monocytogenes , animal pathogenic bacteria , diagnostic techniques , food pathogens , pathogen identification , polymerase chain reaction , quantitative analysis
Source:
Analytical Biochemistry 2013 7 16 v.442 no.1
Language:
English
Year:
2013
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
File:
Download [PDF File]
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.