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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION AND TYPING OF FOOD-BORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples

Authors
item Suo, Biao -
item HE, YIPING
item IRWIN, PETER
item GEHRING, ANDREW

Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2013
Publication Date: May 24, 2013
Repository URL: http://handle.nal.usda.gov/10113/57876
Citation: Suo, B., He, Y., Irwin, P.L., Gehring, A.G. 2013. Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples. Journal of Food Analytical Methods. 6:1477-1484.

Interpretive Summary: Foodborne illness remains a serious public health issue associated with substantial economic cost. To improve food safety and prevent human infections due to ingesting contaminated food, effective detection of a wide range of microbial pathogens in food samples is needed. Previously, we used advanced molecular techniques to design and develop a DNA microarray assay, which was able to simultaneously screen up to 12 independent samples and identify four major foodborne pathogens, including Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Campylobacter jejuni. To improve the performance and minimize the cost of the assay, in this study, we have methodically examined and compared a number of protocols and procedures in the microarray design and fabrication, DNA amplification and labeling, and data acquisition. Through systematical evaluation, optimized conditions for the microarray assay, as well as improvements in pathogen detection are reported. Application of the optimized microarray assay for the detection and genotyping of E. coli O157:H7 in fresh meat samples was demonstrated. Use of the assay to detect pathogens may prevent contaminated food from reaching the consumer.

Technical Abstract: DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and content for foodborne pathogen detection was developed through the systematic evaluation of spotting buffers, probe lengths, scanning conditions, and nucleic acid amplification and labeling strategies. Briefly, by robotic contact printing, a spotting solution of 50% DMSO produced uniform and high-quality spots on UltraGAPS glass slides coated with Amino Propyl Silane. The use of 60% photomultiplier tube (PMT) gain in scanning of ~70-mer oligonucleotide probes resulted in strong signals and low background. For sample preparation, multiplex PCR amplification coupled with fluorescent labeling of DNA using the Klenow fragment and random hexamers achieved higher specificity than whole genome random amplification. To minimize the cost of the assay, the quantities of probes, Klenow fragment, and Cy5 were substantially reduced in each assay without noticeably affecting the detection efficiency. Applying the optimized microarray assay to 26 fresh meat samples, we found 3 different isolates of E. coli O157:H7 in 4 individual packages, demonstrating that the assay has a great potential for identifying and genotyping multiple pathogens in a real food system.

Last Modified: 9/29/2014
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