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| Status |
Public on Apr 02, 2008 |
| Title |
Detection of virulence and antibiotic resistance gene families |
| Platform organisms |
Staphylococcus aureus subsp. aureus Mu50; Escherichia coli CFT073; Enterococcus faecalis V583; Escherichia coli str. K-12 substr. MG1655 |
| Sample organisms |
Staphylococcus aureus; Salmonella enterica; Staphylococcus saprophyticus; unidentified; Escherichia coli O157:H7 str. EDL933; Staphylococcus aureus subsp. aureus Mu50; Escherichia coli CFT073; Enterococcus faecalis V583; Streptococcus pyogenes MGAS5005 |
| Experiment type |
Other
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| Summary |
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
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| Overall design |
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.
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| Contributor(s) |
Jaing C, Gardner S, McLoughlin K, Mulakken N, Williams P, Slezak T |
| Citation(s) |
18478124 |
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| Submission date |
Apr 01, 2008 |
| Last update date |
Mar 19, 2012 |
| Contact name |
Crystal Jaing |
| Organization name |
Lawrence Livermore National Laboratory
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| Street address |
7000 East Ave.
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| City |
Livermore |
| State/province |
CA |
| ZIP/Postal code |
94550 |
| Country |
USA |
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| Platforms (1) |
| GPL6656 |
LLNL Virulence Mechanism Array, version 2A |
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| Samples (12)
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| GSM277643 |
E.faecalis genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278466 |
E.coli CFT073 genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278467 |
E.coli O157:H7 EDL 933 genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278468 |
S.enterica serovar paratyphi genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278469 |
S.pyogenes genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278470 |
S.saprophyticus genomic DNA hybridization to LLNL virulence mechanism array v2A |
| GSM278471 |
LLNL virulence mechanism array version 2A LOD experiment: background only |
| GSM278472 |
LLNL virulence mechanism array version 2A LOD experiment: 0.31 fg S.aureus DNA |
| GSM278473 |
LLNL virulence mechanism array version 2A LOD experiment: 3.1 fg S.aureus DNA |
| GSM278474 |
LLNL virulence mechanism array version 2A LOD experiment: 31 fg S.aureus DNA |
| GSM278475 |
LLNL virulence mechanism array version 2A LOD experiment: 310 fg S.aureus DNA |
| GSM278476 |
LLNL virulence mechanism array version 2A LOD experiment: 3.1 pg S.aureus DNA |
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| Relations |
| BioProject |
PRJNA107095 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE11010_RAW.tar |
75.0 Mb |
(http)(custom) |
TAR (of PAIR) |
| Processed data included within Sample table |
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