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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

Hybridization of E. coli CFT073 and E. faecalis pure genomic DNA to NimbleGen virulence array.A. 4μg of Cy3-labeled E. coli CFT073 or B. E. faecalis DNA were hybridized to the array and the log2 intensity vs probe complement ΔG was plotted. Random control probes, E. coli CFT073, E. coli K12, E. faecalis and S. aureus strain –specific probes are shown in red, yellow, green, cyan and purple colors. The probes that are specific to virulence genes present in the target strain have much higher signal intensities than the random control probes and probes specific to the other three organisms. The detection threshold was set as median + 4 SD.
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pone-0002163-g004: Hybridization of E. coli CFT073 and E. faecalis pure genomic DNA to NimbleGen virulence array.A. 4μg of Cy3-labeled E. coli CFT073 or B. E. faecalis DNA were hybridized to the array and the log2 intensity vs probe complement ΔG was plotted. Random control probes, E. coli CFT073, E. coli K12, E. faecalis and S. aureus strain –specific probes are shown in red, yellow, green, cyan and purple colors. The probes that are specific to virulence genes present in the target strain have much higher signal intensities than the random control probes and probes specific to the other three organisms. The detection threshold was set as median + 4 SD.

Mentions: To assess the ability of the NimbleGen array to reliably identify a target organism of known genome sequence, we performed BLAST searches for all target probe sequences against the four genomes (sequences derived from GenBank), and selected subsets of probes that had a full length perfect match to one genome, and no perfect match longer than 16 nt to any of the other 3 genomes. We refer to these probes as strain-specific probes. We performed hybridizations with purified genomic DNA from either E. coli CFT073, or E. faecalis or E. coli K12. Figure 4 shows log2 intensities plotted against ΔGcomplement for the strain-specific probes for the E. coli CFT073 and E. faecalis hybridizations. The dotted blue line in each plot is the detection threshold for each array representing the median+4 SD of the negative controls (random sequence probes).


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)

Hybridization of E. coli CFT073 and E. faecalis pure genomic DNA to NimbleGen virulence array.A. 4μg of Cy3-labeled E. coli CFT073 or B. E. faecalis DNA were hybridized to the array and the log2 intensity vs probe complement ΔG was plotted. Random control probes, E. coli CFT073, E. coli K12, E. faecalis and S. aureus strain –specific probes are shown in red, yellow, green, cyan and purple colors. The probes that are specific to virulence genes present in the target strain have much higher signal intensities than the random control probes and probes specific to the other three organisms. The detection threshold was set as median + 4 SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002163-g004: Hybridization of E. coli CFT073 and E. faecalis pure genomic DNA to NimbleGen virulence array.A. 4μg of Cy3-labeled E. coli CFT073 or B. E. faecalis DNA were hybridized to the array and the log2 intensity vs probe complement ΔG was plotted. Random control probes, E. coli CFT073, E. coli K12, E. faecalis and S. aureus strain –specific probes are shown in red, yellow, green, cyan and purple colors. The probes that are specific to virulence genes present in the target strain have much higher signal intensities than the random control probes and probes specific to the other three organisms. The detection threshold was set as median + 4 SD.
Mentions: To assess the ability of the NimbleGen array to reliably identify a target organism of known genome sequence, we performed BLAST searches for all target probe sequences against the four genomes (sequences derived from GenBank), and selected subsets of probes that had a full length perfect match to one genome, and no perfect match longer than 16 nt to any of the other 3 genomes. We refer to these probes as strain-specific probes. We performed hybridizations with purified genomic DNA from either E. coli CFT073, or E. faecalis or E. coli K12. Figure 4 shows log2 intensities plotted against ΔGcomplement for the strain-specific probes for the E. coli CFT073 and E. faecalis hybridizations. The dotted blue line in each plot is the detection threshold for each array representing the median+4 SD of the negative controls (random sequence probes).

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