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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

Detection of virulence genes from S. aureus spiked into BioWatch aerosol filter samples.0.31 fg, 3.1 fg, 31 fg, 310 fg and 3.1 pg of S. aureus DNA were spiked into 10 ng of extracted BioWatch aerosol DNA samples. Aerosol sample alone or the mixed DNA samples were amplified, labeled and hybridized. Plots show log2 intensity of probes vs ΔG complement. Array hybridized with aerosol sample only is shown in Figure 7A and the arrays hybridized with 0.31 fg to 3.1 pg spiked in S. aureus are shown in Figures 7B-9F. 100% of the probes specific to virulence genes in S. aureus were detected in 3.1 fg and above. 36% of the probes were detected at 0.31 fg of S. aureus.
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pone-0002163-g007: Detection of virulence genes from S. aureus spiked into BioWatch aerosol filter samples.0.31 fg, 3.1 fg, 31 fg, 310 fg and 3.1 pg of S. aureus DNA were spiked into 10 ng of extracted BioWatch aerosol DNA samples. Aerosol sample alone or the mixed DNA samples were amplified, labeled and hybridized. Plots show log2 intensity of probes vs ΔG complement. Array hybridized with aerosol sample only is shown in Figure 7A and the arrays hybridized with 0.31 fg to 3.1 pg spiked in S. aureus are shown in Figures 7B-9F. 100% of the probes specific to virulence genes in S. aureus were detected in 3.1 fg and above. 36% of the probes were detected at 0.31 fg of S. aureus.

Mentions: Figure 7 shows the intensity of strain-specific probes versus ΔGcomplement for arrays hybridized to each of the six samples. In the unspiked aerosol background DNA, we found only a few probes with signals barely above the detection threshold; therefore we expect that the signal seen in the spiked samples is mostly or entirely due to the added S. aureus DNA. With 0.31 fg of S. aureus DNA, we observe about 36% of S. aureus–specific probes with signals above the threshold. The detectable probes cover about 37% of the targeted virulence gene orthologs. This level of detection was reproducibly observed in multiple experiments. With 3.1 fg, we see that 100% of the S. aureus specific probes were above the detection threshold. With 31, 310 or 3100 fg, virtually all of the S. aureus specific probes were saturated, with intensities within a factor of two below the maximum possible intensity.


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)

Detection of virulence genes from S. aureus spiked into BioWatch aerosol filter samples.0.31 fg, 3.1 fg, 31 fg, 310 fg and 3.1 pg of S. aureus DNA were spiked into 10 ng of extracted BioWatch aerosol DNA samples. Aerosol sample alone or the mixed DNA samples were amplified, labeled and hybridized. Plots show log2 intensity of probes vs ΔG complement. Array hybridized with aerosol sample only is shown in Figure 7A and the arrays hybridized with 0.31 fg to 3.1 pg spiked in S. aureus are shown in Figures 7B-9F. 100% of the probes specific to virulence genes in S. aureus were detected in 3.1 fg and above. 36% of the probes were detected at 0.31 fg of S. aureus.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002163-g007: Detection of virulence genes from S. aureus spiked into BioWatch aerosol filter samples.0.31 fg, 3.1 fg, 31 fg, 310 fg and 3.1 pg of S. aureus DNA were spiked into 10 ng of extracted BioWatch aerosol DNA samples. Aerosol sample alone or the mixed DNA samples were amplified, labeled and hybridized. Plots show log2 intensity of probes vs ΔG complement. Array hybridized with aerosol sample only is shown in Figure 7A and the arrays hybridized with 0.31 fg to 3.1 pg spiked in S. aureus are shown in Figures 7B-9F. 100% of the probes specific to virulence genes in S. aureus were detected in 3.1 fg and above. 36% of the probes were detected at 0.31 fg of S. aureus.
Mentions: Figure 7 shows the intensity of strain-specific probes versus ΔGcomplement for arrays hybridized to each of the six samples. In the unspiked aerosol background DNA, we found only a few probes with signals barely above the detection threshold; therefore we expect that the signal seen in the spiked samples is mostly or entirely due to the added S. aureus DNA. With 0.31 fg of S. aureus DNA, we observe about 36% of S. aureus–specific probes with signals above the threshold. The detectable probes cover about 37% of the targeted virulence gene orthologs. This level of detection was reproducibly observed in multiple experiments. With 3.1 fg, we see that 100% of the S. aureus specific probes were above the detection threshold. With 31, 310 or 3100 fg, virtually all of the S. aureus specific probes were saturated, with intensities within a factor of two below the maximum possible intensity.

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