E-GEOD-11010 - Detection of virulence and antibiotic resistance gene families

Status
Released on 2 April 2008, last updated on 27 March 2012
Organism
Enterococcus faecalis V583, Escherichia coli CFT073, Escherichia coli O157:H7 EDL933, Salmonella enterica, Staphylococcus aureus, Staphylococcus aureus subsp. aureus Mu50, Staphylococcus saprophyticus subsp. saprophyticus, Streptococcus pyogenes MGAS5005, unidentified
Samples (12)
Array (1)
Protocols (13)
Description
Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known and novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. Keywords: detection, pathogen, virulence mechanism In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.
Experiment type
other 
Contacts
Crystal Jaing <geo@ncbi.nlm.nih.gov>, Kevin McLoughlin, Nisha Mulakken, Peter Williams, Shea Gardner, Tom Slezak
Citation
A functional gene array for detection of bacterial virulence elements. Jaing C, Gardner S, McLoughlin K, Mulakken N, Alegria-Hartman M, Banda P, Williams P, Gu P, Wagner M, Manohar C, Slezak T. , Europe PMC 18478124
MIAME
PlatformsProtocolsFactorsProcessedRaw
Files
Investigation descriptionE-GEOD-11010.idf.txt
Sample and data relationshipE-GEOD-11010.sdrf.txt
Raw data (1)E-GEOD-11010.raw.1.zip
Processed data (1)E-GEOD-11010.processed.1.zip
Array designA-GEOD-6656.adf.txt
Links