title: Adaptation and Validation of E-Probe Diagnostic Nucleic Acid Analysis for Detection of Escherichia coli O157:H7 in Metagenomic Data from Complex Food Matrices.
abstract: The Centers for Disease Control and Prevention recently emphasized the need for enhanced technologies to use in investigations of outbreaks of foodborne illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA) was adapted and validated as a tool for the rapid, effective identification and characterization of multiple pathogens in a food matrix. In EDNA, unassembled next generation sequencing data sets from food sample metagenomes are queried using pathogen-specific sequences known as electronic probes (e-probes). In this study, the query of mock sequence databases demonstrated the potential of EDNA for the detection of foodborne pathogens. The method was then validated using next generation sequencing data sets created by sequencing the metagenome of alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the negative control sample indicated the need for additional filtration of the e-probes to enhance specificity. There was no significant difference in the ability of an e-probe to detect the target pathogen based upon the length of the probe set oligonucleotides. The results from the queries of the sample database using E. coli e-probe sets were significantly different from those obtained using random decoy probe sets and exhibited 100% precision. The results support the use of EDNA as a rapid response methodology in foodborne outbreaks and investigations for establishing comprehensive microbial profiles of complex food samples.
authors.affiliation: Department of Entomology and Plant Pathology, 127 NRC, Oklahoma State University, Stillwater, Oklahoma 74078, USA. trenna.bladgen@okstate.edu.
authors.forename: Trenna
authors.initials: T
authors.lastname: Blagden
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Room 139, Building 1301, 1301 Ditto Avenue, Fort Detrick, Maryland 21702, USA.
authors.forename: William
authors.initials: W
authors.lastname: Schneider
authors.affiliation: Department of Biochemistry and Molecular Biology, 246 NRC, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Melcher
authors.affiliation: Department of Entomology and Plant Pathology, 127 NRC, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Daniels
authors.affiliation: Department of Entomology and Plant Pathology, 127 NRC, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Fletcher
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis; D005517:Foodborne Diseases / Q000382:microbiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000455:Medicago sativa / Q000382:microbiology*; D056186:Metagenomics / Q000379:methods*; D014481:United States; D014675:Vegetables / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial