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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

Langmuir isotherm fit of adjusted ΔG vs median log intensity of one array.A linear combination of the three free energies (ΔGcomplement, ΔGhomodimer and ΔGhairpin) which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA.
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pone-0002163-g003: Langmuir isotherm fit of adjusted ΔG vs median log intensity of one array.A linear combination of the three free energies (ΔGcomplement, ΔGhomodimer and ΔGhairpin) which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA.

Mentions: We observed that the relationship of log intensity to thermodynamic parameters such as ΔGcomplement is nonlinear, and shows evidence of chemical saturation for the most sensitive probes. In order to incorporate saturation into our probe response model and find the combination of thermodynamic parameters that was the best predictor of probe sensitivity, we fit our data to a Langmuir isotherm curve [12] (Figure 3), parameterized by the ΔGcomplement, ΔGhomodimer, and ΔGhairpin as follows:We performed a nonlinear least squares fit to data from eight microarrays, each hybridized to 1–5 μg of DNA from one of the four target species, to fit values for the parameters a0 through a5. We determined that a linear combination of the three free energies which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA. The ΔGadjusted is defined as:


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)

Langmuir isotherm fit of adjusted ΔG vs median log intensity of one array.A linear combination of the three free energies (ΔGcomplement, ΔGhomodimer and ΔGhairpin) which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002163-g003: Langmuir isotherm fit of adjusted ΔG vs median log intensity of one array.A linear combination of the three free energies (ΔGcomplement, ΔGhomodimer and ΔGhairpin) which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA.
Mentions: We observed that the relationship of log intensity to thermodynamic parameters such as ΔGcomplement is nonlinear, and shows evidence of chemical saturation for the most sensitive probes. In order to incorporate saturation into our probe response model and find the combination of thermodynamic parameters that was the best predictor of probe sensitivity, we fit our data to a Langmuir isotherm curve [12] (Figure 3), parameterized by the ΔGcomplement, ΔGhomodimer, and ΔGhairpin as follows:We performed a nonlinear least squares fit to data from eight microarrays, each hybridized to 1–5 μg of DNA from one of the four target species, to fit values for the parameters a0 through a5. We determined that a linear combination of the three free energies which we term “ΔGadjusted” was the best predictor of hybridization intensity for probes complementary to the target DNA. The ΔGadjusted is defined as:

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