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Title: TIME-RESOLVED FLUORESCENCE DETECTION OF TOXINS AND PATHOGENIC BACTERIA IN FOODS

Author
item Tu, Shu I
item Golden, Marsha
item Paoli, George
item GORE, MITCHELL - POLYSCIENCES
item Gehring, Andrew

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2004
Publication Date: 12/12/2004
Citation: Tu, S., Golden, M., Paoli, G., Gore, M., Gehring, A.G. 2004. Time-resolved fluorescence detection of toxins and pathogenic bacteria in foods. Journal of Rapid Methods and Automation in Microbiology. 12. pp. 247-258.

Interpretive Summary: Contamination of viable pathogenic bacteria in foods may lead to serious public health concerns. Among the disease-causing bacteria, Enterohemorrhagic Escherichia coli (EHEC) are known to produce Shiga like toxins (SLT) that may cause bloody diarrhea and sometime lead to life threatening acute kidney failure. To minimize possible outbreak of food poisoning by pathogenic bacteria, sensitive and rapid detection techniques for the bacteria and the toxins are needed to call for proper treatments to intervene further distribution of contaminated foods. Current available methods, e.g., tissue culture test, for detecting SLTs are expensive and time consuming and have low sensitivity. In this work, we developed a new process to detect the bacteria and SLTs from the same samples. The method involved the applications of specific antibodies to capture the bacteria and SLTs and the capture signals are then amplified by very sensitive fluorescence detection. The developed method is about 200 times more sensitive than the tissue culture method in detecting SLTs. The information is useful for researcher and/or engineers to design a process to detect specific pathogens and their toxins in foods.

Technical Abstract: Immunomagnetic beads specific to Shiga-Like Toxin 1 (SLT-I) and Shiga-Like Toxin 2 (SLT-II) were applied to capture and concentrate the toxins in solution. The captured toxins were further labeled by Europium (Eu) -tagged anti-toxin antibody prior to detection by a time-resolved fluorescence (TRF) reader. The sensitivity of this method to the toxins was estimated as between 5 and 50 pg/mL. The toxins produced by Escherichia coli O157:H7 spiked in ground beef could be detected. However, toxin detection required much longer enrichment time (overnight) then that for the bacteria (~ 4 h) detection from ground beef spiked with E. coli O157:H7 (1 CFU/g). To increase the concentration of SLTs for TRF detection, ofloxacin was added to enhance the cellular production of SLTs and bacterial protein extraction reagent (B-PER) was used to release cellular SLTs. These approaches allowed the detection of SLTs associated with 1 CFU of E. coli O157:H7 harvested at post-stationary growth phase. The production of the toxins by non-O157 Enterohemorrhagic E. coli (EHEC) spiked in ground beef could be detected by the same method with a similar sensitivity. The method allowed the detection of EHEC and SLTs from the same food samples using the same experimental procedure and instruments.