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Basic concepts of microarrays and potential applications in clinical microbiology - PubMed

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Review

Basic concepts of microarrays and potential applications in clinical microbiology

Melissa B Miller et al. Clin Microbiol Rev. 2009 Oct.

Abstract

The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles.

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Figures

FIG. 1.
FIG. 1.

Microarray publications. The number of primary manuscripts published using microarray technology (bars) and the number of microarray publications that have infectious disease and/or microbiology applications (line) are depicted.

FIG. 2.
FIG. 2.

Workflow summary of printed microarrays. Probes are PCR amplified (or oligonucleotides are synthesized) and subsequently spotted onto a glass slide. In this example, two samples to be compared undergo RNA extraction, cDNA production, and differential fluorescent labeling. Hybridization of labeled target nucleic acids to the probe array allows fluorescent scanning to provide data for analysis. (Adapted from reference [Fig. 1A, © Springer-Verlag 2006] with kind permission from Springer Science and Business Media.)

FIG. 3.
FIG. 3.

Affymetrix GeneChip oligonucleotide microarray. (Top) Photolithography. UV light is passed through a lithographic mask that acts as a filter to either transmit or block the light from the chemically protected microarray surface (wafer). The sequential application of specific lithographic masks determines the order of sequence synthesis on the wafer surface. (Bottom) Chemical synthesis cycle. UV light removes the protecting groups (squares) from the array surface, allowing the addition of a single protected nucleotide as it is washed over the microarray. Sequential rounds of light deprotection, changes in the filtering patterns of the masks, and single nucleotide additions form microarray features with specific 25-bp probes. (Adapted from reference with permission of the publisher [copyright Elsevier Inc. 2006].)

FIG. 4.
FIG. 4.

Roche NimbleGen oligonucleotide microarray. Maskless array synthesizer technology is depicted, which utilizes a digital micromirror device (DMD) to create virtual masks. The DMD forms the pattern of UV light needed to direct the specific nucleic acid addition during photo-mediated synthesis. UV light removes the photolabile protecting group (circles) from the microarray surface, allowing the addition of a single protected nucleotide to the growing oligonucleotide chain. The cycling of DMD filtering, light deprotection, and nucleotide addition creates oligonucleotide features 60 to 100 bp in length on the NimbleGen microarray. (Courtesy of Roche NimbleGen [copyright Roche NimbleGen, Inc.].)

FIG. 5.
FIG. 5.

Agilent oligonucleotide microarray. (A) Noncontact inkjet printing technology delivers a small and accurate volume (picoliters) of nucleotides to the first layer on the microarray surface. (B) Repeated rounds of base-by-base printing extend the length of specific oligonucleotide probes. (C) Close-up of growing oligonucleotide chain with a base being added. (D) The final product is a 60-mer in situ-synthesized probe as a feature on a microarray containing thousands of specifically synthesized probes. (Images courtesy of Agilent Technologies.)

FIG. 6.
FIG. 6.

Illumina BeadArray. The SAM contains 96 1.4-mm fiber-optic bundles (bottom left). Each bundle is an individual array consisting of 50,000 5-μm fiber-optic strands, each of which is chemically etched to create a microwell for a single bead (top left). The Sentrix BeadChips can assay 1 to 16 samples at a time on a silicon slide (bottom right) that has been processed to provide microwells for individual beads (top right). Both BeadArray platforms rely on 3-μm silica beads that randomly self-assemble (center). (Adapted from reference with permission of the publisher. © 2009 BioTechniques.)

FIG. 7.
FIG. 7.

Electronic microarray. (A) A positive electric current is applied to test sites, facilitating the active movement and concentration of negatively charged DNA probes to the activated locations. (B) Once the first probe is bound to its targeted location(s) by streptavidin-biotin bonds, the test site(s) can be deactivated, and current can be applied to a different test site. This process is repeated until all the probes are arrayed. (C) Nanogen's RVA ASR. Upon application of the probes to targeted test sites, extracted and amplified nucleic acids from a respiratory sample passively hybridize to the microarray surface. If hybridization occurs, secondary probes that are specific for the target and that contain a nonspecific detector sequence will bind. Secondary fluorescent detector oligonucleotides are used to measure positive hybridization reactions. Multiple probes can be used per site when multiple fluorophores are incorporated. P1, parainfluenza virus type 1; P2, parainfluenza virus type 2; P3, parainfluenza virus type 3; FB, influenza B virus; FA, influenza A virus; RSV, respiratory syncytial virus; BKGD, background. (Images courtesy of Nanogen.)

FIG. 8.
FIG. 8.

Suspension bead array. (A) Microspheres 5.6 μm in diameter are filled with different relative concentrations of an infrared dye and a red dye to create 100 beads, each with a unique spectral identity. (B) Potential targets are amplified using a biotinylated primer and then denatured and hybridized to microspheres tagged with target-specific sequence probes. Probe-target hybridization is measured using a streptavidin-bound green fluorophore. (C) Flow cytometry is used to analyze the microsphere suspension. A red laser is used to determine the spectral identity of the bead and, therefore, the probe being analyzed. The reporter fluorochrome is excited by a green laser, which quantifies the probe-target reaction on the microsphere surface. (Panels A and C courtesy of Luminex Corporation; panel B adapted from reference with permission from Elsevier [copyright Elsevier Inc. 2006].)

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References

    1. Akhras, M. S., S. Thiyagarajan, A. C. Villablanca, R. W. Davis, P. Nyren, and N. Pourmand. 2007. PathogenMip assay: a multiplex pathogen detection assay. PLoS ONE 2:e223. - PMC - PubMed
    1. Albert, T. J., D. Dailidiene, G. Dailide, J. E. Norton, A. Kalia, T. A. Richmond, M. Molla, J. Singh, R. D. Green, and D. E. Berg. 2005. Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nat. Methods 2:951-953. - PubMed
    1. Albrecht, V., A. Chevallier, V. Magnone, P. Barbry, F. Vandenbos, A. Bongain, J. C. Lefebvre, and V. Giordanengo. 2006. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays. J. Virol. Methods 137:236-244. - PubMed
    1. Anjum, M. F., M. Mafura, P. Slickers, K. Ballmer, P. Kuhnert, M. J. Woodward, and R. Ehricht. 2007. Pathotyping Escherichia coli by using miniaturized DNA microarrays. Appl. Environ. Microbiol. 73:5692-5697. - PMC - PubMed
    1. Anthony, R. M., T. J. Brown, and G. L. French. 2000. Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array. J. Clin. Microbiol. 38:781-788. - PMC - PubMed

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