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Optimization and Application of a Custom Microarray for the Detection and Genotyping of E. coli O157:H7 in Fresh Meat Samples

Permanent URL:
http://handle.nal.usda.gov/10113/57876
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% dimethylsulfoxide produced uniform and high-quality spots on UltraGAPS glass slides coated with aminopropyl silane. The use of 60% photomultiplier tube gain in scanning ∼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 noticeablyaffecting the detection efficiency. Applying the optimized microarray assay to 26 fresh meat samples, three different isolates of Escherichia coli O157:H7 were found in four individual packages, demonstrating that the assay has a great potential for identifying and genotyping multiple pathogens in a real food system.
Author(s):
Biao Suo , Yiping He , Peter Irwin , Andrew Gehring
Subject(s):
DNA , Escherichia coli O157 , bacterial contamination , buffers , dimethyl sulfoxide , fluorescent labeling , food contamination , food pathogens , genome , genotyping , microarray technology , microbial detection , oligonucleotide probes , polymerase chain reaction , raw meat , sampling
Source:
Food analytical methods 2013 5 28 v.6
Language:
English
Year:
2013
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
File:
Download [PDF File]
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.