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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

Effect of the length of perfect match (PM) sequence on the hybridization of target and probe. 2, 3, 6, 10, 15 and 20 mismatches were randomly created.Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.
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pone-0002163-g006: Effect of the length of perfect match (PM) sequence on the hybridization of target and probe. 2, 3, 6, 10, 15 and 20 mismatches were randomly created.Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.

Mentions: When probes contained multiple mismatches to the genome of the hybridized strain, we found that the reduction in intensity depended not only on the number of mismatched bases, but also on the length of the longest PM sequence between mismatches. Consequently, longer probes tend to be more tolerant of mismatches. The relationship between the number of MM bases, the longest PM region length, and the reduction in intensity relative to the cognate PM probe is shown in Figure 6. The graph shows the mean MM/PM intensity ratios averaged over 60 PM probes and the corresponding random MM probes. The random MM probes were generated from the PM probe sequences by selecting 2, 3, 6, 10, 15 or 20 random positions in the PM probe, and creating single mismatches at each position. Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.


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)

Effect of the length of perfect match (PM) sequence on the hybridization of target and probe. 2, 3, 6, 10, 15 and 20 mismatches were randomly created.Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002163-g006: Effect of the length of perfect match (PM) sequence on the hybridization of target and probe. 2, 3, 6, 10, 15 and 20 mismatches were randomly created.Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.
Mentions: When probes contained multiple mismatches to the genome of the hybridized strain, we found that the reduction in intensity depended not only on the number of mismatched bases, but also on the length of the longest PM sequence between mismatches. Consequently, longer probes tend to be more tolerant of mismatches. The relationship between the number of MM bases, the longest PM region length, and the reduction in intensity relative to the cognate PM probe is shown in Figure 6. The graph shows the mean MM/PM intensity ratios averaged over 60 PM probes and the corresponding random MM probes. The random MM probes were generated from the PM probe sequences by selecting 2, 3, 6, 10, 15 or 20 random positions in the PM probe, and creating single mismatches at each position. Intensity ratios were averaged over 10 arrays for the 60 sets of PM and MM probes, and are plotted here on a log scale against the length of the longest PM region.

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