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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY AND GENOMICS OF FOODBORNE PATHOGENS Title: Identification of Escherichia coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

Authors
item Quiñones, Beatriz
item Swimley, Michelle
item Taylor, Amber -
item Dawson, Erica -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 16, 2010
Publication Date: June 1, 2011
Repository URL: http://www.libertononline.com/doi/pdfplus/10.1089/fpd.2010.0753
Citation: Quinones, B., Swimley, M.S., Taylor, A.W., Dawson, E.D. 2011. Identification of Escherichia coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays. Foodborne Pathogens and Disease. 8(6):705-7011.

Interpretive Summary: Shiga toxin-producing Escherichia coli is an enteric pathogen known to cause human gastrointestinal illnesses with clinical spectra, ranging from bloody diarrhea and hemorrhagic colitis to life-threatening hemolytic-uremic syndrome. In the recent years, most risk management efforts have focused on identifying the presence and distribution of E. coli O157:H7 in foods as part of imperative constituents of food safety programs. Thus, the O157 serotype is considered to be the most-commonly reported serotype linked to outbreaks in North America. The rise in foodborne-related outbreaks of E. coli O157 has heightened the importance of developing improved methods to rapidly detect and characterize virulent strains. Established culturing methods for the identification of pathogenic E. coli can be labor-intensive and time-consuming and assess phenotypic and genotypic markers by screening only a small number of determinants. Molecular-based technologies, such as DNA microarrays, offer a viable alternative to screen simultaneously multiple markers. One challenge of using the DNA microarray platform for pathogen detection has been developing cost effective as well as sensitive procedures that would indicate the positive signals on the array. Fluorescent assays are commonly-used methods for microarray-based detection of pathogens. However, limitations of these assays are that labeling of target DNA can be inconsistent and highly variable and utilize expensive and non-portable scanners for data acquisition and analysis. Other non-fluorescent, colorimetric assays employ the use of unstable reagents that require temperature-controlled environments and that have variable development times, leading to overexposure in the signal detection. Improved procedures for pathogen surveillance are thus needed with sufficient sensitivity, cost-effectiveness, and suitability for routine testing. To implement the use of better detection methods for categorizing E. coli O157 strains, the present study evaluated ampliPHOX, a novel and innovative colorimetric procedure that is based on light-initiated signal amplification through polymerization. Our findings demonstrated that photopolymerization is a simple and rapid method for the accurate genotyping of E. coli O157 strains with DNA microarrays.

Technical Abstract: Shiga toxin-producing Escherichia coli O157:H7 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, while average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated the detection sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

Last Modified: 10/30/2014
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