title: Identification of Escherichia coli O157 by using a novel colorimetric detection method with DNA microarrays.
abstract: Shiga toxin-producing Escherichia coli O157 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, whereas 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 that the 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.
authors.affiliation: Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, USA. beatriz.quinones@ars.usda.gov
authors.forename: Beatriz
authors.initials: B
authors.lastname: Quiñones
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Swimley
authors.forename: Amber W
authors.initials: AW
authors.lastname: Taylor
authors.forename: Erica D
authors.initials: ED
authors.lastname: Dawson
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D003124:Colorimetry; D019453:Escherichia coli O157 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005517:Foodborne Diseases / Q000517:prevention & control; D005798:Genes, Bacterial; D005838:Genotype; D007202:Indicators and Reagents / Q000737:chemistry; D058889:Molecular Typing / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D058105:Polymerization; D022622:Shiga Toxin 1 / Q000235:genetics / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D000637:Transaminases / Q000235:genetics / Q000378:metabolism; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D037521:Virulence Factors; C073819:eaeA protein, E coli; D001710:Biotin; D019809:Streptavidin; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase