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United States Department of Agriculture

Agricultural Research Service

Title: Luminescent Methods to Detect Viable and Total Targeted Escherichia Coli 0157:h7 Spiked in Ground Beef

Authors
item Tu, Shu-I
item Uknalis, Joseph
item Yamashoji, Shiro - NIKKEN BIOMED LAB JAPAN
item Gehring, Andrew
item Irwin, Peter

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 21, 2005
Publication Date: June 16, 2005
Citation: Tu, S., Uknalis, J., Yamashoji, S., Gehring, A.G., Irwin, P.L. 2005. Luminescent methods to detect viable and total targeted escherichia coli 0157:h7 spiked in ground beef. Journal of Rapid Methods and Automation in Microbiology. 2005. 13. pp. 57-70.

Interpretive Summary: Contamination of foods by pathogenic bacteria, e.g., E coli O157:H7 may lead to serious public health concerns. To minimize possible outbreaks of food poisoning, sensitive, rapid and specific pathogen detection techniques are needed. In this work, we developed a new luminescence process to detect E. coli O157:H7 in ground beef. This process involved detecting the presence of the pathogen and followed by testing the viability of the organism. The newly developed process allows the use of the same instrumentation and chemical principles for both steps. The obtained result will be useful for researchers/engineers in designing cost-effective detection procedures that can generate multifaceted information of the pathogen using a single detection platform.

Technical Abstract: The presence of E. coli O157:H7 in food is a serious public health concern. Pathogens which occur in meat are not necessarily all viable. Sensitive and efficient methods are needed to detect the presence of viable E. coli O157:H7 in ground beef. In this work, known quantities of the E. coli were used to spike meat samples. The contaminated samples were incubated in EC + novobiocin growth media at 37 degrees C for 5 h. The bacteria were captured by magnetic beads coated with anti-E. coli O157 antibodies which were then exposed to a second anti-E. coli O157 antibody labeled with peroxidase. Alternatively, the captured bacteria were treated with membrane permeable electron-transfer shuttering reagent such as menadione to oxidize the cellular NAD(P)H. The enzyme activity of labeled peroxidase and the level of cellular content of NAD(P)H were then measured by luminol-linked luminescence. Treating the E. coli O157:H7 with a bacterial protein extraction reagent (B-PER), diminished the luminescence associated with the cellular NAD(P)H but not with the sandwiched peroxidase labels. Thus, the NAD(P)H-linked luminescence, but not that associated with the sandwiched complexes, was related to the viability of the E. coli. The results indicate that the combination of the both luminescence methods, in a 96 well microplate format, could be used to detect the viable E. coli in ground beef.

Last Modified: 12/18/2014
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