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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

Log2 intensity vs probe length, predicted melting temperature Tm, and predicted complement ΔG for selected probes in an array hybridized with E. coli CFT073 genomic DNA.Probes specific for E. coli sequences are plotted in green; probes specific for E. faecalis are in red.
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pone-0002163-g002: Log2 intensity vs probe length, predicted melting temperature Tm, and predicted complement ΔG for selected probes in an array hybridized with E. coli CFT073 genomic DNA.Probes specific for E. coli sequences are plotted in green; probes specific for E. faecalis are in red.

Mentions: Figure 2 shows log signal intensities for probes targeting a typical gene family, in which DNA complementary to one set of probes (those for E. coli CFT073) was present in the hybridization mix, while DNA for another set of probes (for E. faecalis) was absent; thus the signal seen for E. faecalis probes is entirely due to non-specific hybridization and other sources of background noise. We found that probes with lengths above 50 nt gave significantly stronger signals, with better differentiation from background, than lengths in the 30 to 45 nt range. The predicted melting temperature and ΔGcomplement are strongly correlated with probe length, but not entirely determined by it. We performed linear regression fits to the log intensity against each of the probe design parameters, and multiple regressions against several combinations of parameters. Of the individual probe parameters we examined, the best predictor of intensity (i.e., the one with the smallest residual variance) was ΔGcomplement; the best multivariate predictor was a combination of ΔGcomplement, ΔGhomodimer, and ΔGhairpin.


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)

Log2 intensity vs probe length, predicted melting temperature Tm, and predicted complement ΔG for selected probes in an array hybridized with E. coli CFT073 genomic DNA.Probes specific for E. coli sequences are plotted in green; probes specific for E. faecalis are in red.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002163-g002: Log2 intensity vs probe length, predicted melting temperature Tm, and predicted complement ΔG for selected probes in an array hybridized with E. coli CFT073 genomic DNA.Probes specific for E. coli sequences are plotted in green; probes specific for E. faecalis are in red.
Mentions: Figure 2 shows log signal intensities for probes targeting a typical gene family, in which DNA complementary to one set of probes (those for E. coli CFT073) was present in the hybridization mix, while DNA for another set of probes (for E. faecalis) was absent; thus the signal seen for E. faecalis probes is entirely due to non-specific hybridization and other sources of background noise. We found that probes with lengths above 50 nt gave significantly stronger signals, with better differentiation from background, than lengths in the 30 to 45 nt range. The predicted melting temperature and ΔGcomplement are strongly correlated with probe length, but not entirely determined by it. We performed linear regression fits to the log intensity against each of the probe design parameters, and multiple regressions against several combinations of parameters. Of the individual probe parameters we examined, the best predictor of intensity (i.e., the one with the smallest residual variance) was ΔGcomplement; the best multivariate predictor was a combination of ΔGcomplement, ΔGhomodimer, and ΔGhairpin.

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