Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/5/2013
Publication Date: 7/16/2013
Citation: Brewster, J.D., Paoli, G. 2013. DNA extraction protocol for rapid PCR detection of pathogenic bacteria. Analytical Biochemistry. 442(1):107-109
Interpretive Summary: Rapid, sensitive assays capable of detecting small numbers of pathogenic bacteria in foods are needed by regulatory inspection personnel and others in the agriculture and food industries working to reduce foodborne illnesses, such as those caused by E. coli O157:H7 and Listeria monocytogenes. Current techniques are not able to detect very low levels of pathogens, so it is necessary to culture samples for a period of time to allow the pathogen to grow and increase in concentration before detection. This enrichment process adds many hours to the assay, among other limitations. ARS has developed a novel filtration process that separates and concentrates bacteria from food samples much more quickly than enrichment. We have reported the combination of this process with a new DNA extraction protocol and a very sensitive detection technique (real-time polymerase chain reaction) to assay low levels of E. coli O157:H7 in ground beef in a few hours. This report describes in detail the development of the rapid DNA extraction protocol, and how it can be optimized for other foods and pathogens.
Technical Abstract: Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organisms in one assay. Development of new approaches for very rapid (2 hr) direct PCR detection without enrichment is hampered by current DNA extraction methods, which exhibit significant differences in extraction efficiency across genera and deliver only a small fraction of the extracted DNA to the PCR assay. We have developed a novel 15 minute direct-lysis method for DNA extraction that provides high DNA recovery for both Gram-negative (E. coli O157:H7) and Gram-positive (Listeria monocytogenes) pathogens and allows the entire DNA extract to be added to a 20 mul reaction without inhibition of the PCR.