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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 - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chemistry, Materials, Earth and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, United States of America. jaing2/at/llnl.gov

ABSTRACT
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 or 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.

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Related in: MedlinePlus

Virulence array probe design process.Candidate probes were generated using Primer3 and Unafold based on Tm, GC content, salt concentration, and minimum free energies of probes. Probes were filtered based on best free energy and duplicate probe sequences were removed. When necessary, additional candidate probes were generated using more relaxed parameters to ensure full coverage. The final set of probes was then downselected to produce a maximum of 30 probes per equivalence group, each capable of detecting multiple target sequences in a given family.
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pone-0002163-g001: Virulence array probe design process.Candidate probes were generated using Primer3 and Unafold based on Tm, GC content, salt concentration, and minimum free energies of probes. Probes were filtered based on best free energy and duplicate probe sequences were removed. When necessary, additional candidate probes were generated using more relaxed parameters to ensure full coverage. The final set of probes was then downselected to produce a maximum of 30 probes per equivalence group, each capable of detecting multiple target sequences in a given family.

Mentions: After selecting and extracting target gene sequences, we designed probes as diagrammed in Figure 1. In summary, we selected probes for a given gene family using a greedy algorithm favoring the most conserved regions of sequences within that family, while ensuring that each target sequence had a minimum number of probes that were complementary to it. More details follow below. Using the most conserved regions enabled coverage of more sequences with fewer probes, and thus detection of more potential families on a single array, than simply tiling probes across each target sequence. We included additional probes for divergent sequences not captured by the conserved probes so that all known orthologs within the 4 genomes could be detected.


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 - PLoS ONE (2008)

Virulence array probe design process.Candidate probes were generated using Primer3 and Unafold based on Tm, GC content, salt concentration, and minimum free energies of probes. Probes were filtered based on best free energy and duplicate probe sequences were removed. When necessary, additional candidate probes were generated using more relaxed parameters to ensure full coverage. The final set of probes was then downselected to produce a maximum of 30 probes per equivalence group, each capable of detecting multiple target sequences in a given family.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367441&req=5

pone-0002163-g001: Virulence array probe design process.Candidate probes were generated using Primer3 and Unafold based on Tm, GC content, salt concentration, and minimum free energies of probes. Probes were filtered based on best free energy and duplicate probe sequences were removed. When necessary, additional candidate probes were generated using more relaxed parameters to ensure full coverage. The final set of probes was then downselected to produce a maximum of 30 probes per equivalence group, each capable of detecting multiple target sequences in a given family.
Mentions: After selecting and extracting target gene sequences, we designed probes as diagrammed in Figure 1. In summary, we selected probes for a given gene family using a greedy algorithm favoring the most conserved regions of sequences within that family, while ensuring that each target sequence had a minimum number of probes that were complementary to it. More details follow below. Using the most conserved regions enabled coverage of more sequences with fewer probes, and thus detection of more potential families on a single array, than simply tiling probes across each target sequence. We included additional probes for divergent sequences not captured by the conserved probes so that all known orthologs within the 4 genomes could be detected.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chemistry, Materials, Earth and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, United States of America. jaing2/at/llnl.gov

ABSTRACT
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 or 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.

Show MeSH
Related in: MedlinePlus