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Chapter 18 : Clinical Microbiology: Looking Ahead

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Abstract:

This chapter provides an overview of new and emerging molecular diagnostic technologies. While not all techniques can be described here, examples of relevant technologies for specimen preparation, PCR, post-PCR detection, and analysis are presented. Several non-PCR methods are discussed. Advances in fluorescent chemistry have facilitated fluorescent detection of nucleic acids or hybridized nucleic acid probes and have enabled technology to advance from traditional end product analysis to real-time monitoring of PCR amplicons within a closed system. This strategy may very well be the most exciting advance for clinical microbiology since the advent of PCR itself. An alternative to fluorescent-dye incorporation, fluorescence resonance energy transfer (FRET) technology, allows the detection and quantitation of specific PCR products through the use of nucleic acid probes. Current limitations of microarray technology include the high costs of instrumentation and disposables. In addition, one's current inability to easily analyze the enormous amount of information potentially available through this technology will present challenges. It is quite possible that peptide nucleic acids (PNAs) will have practical applications in molecular diagnostics. Advances in nonisotopic, non-gel-based detection and identification of PCR products may allow for cost-effective integration into the clinical microbiology workplace. Microbiologists must responsibly prepare molecular algorithms to include the ability to identify new pathogens or new presentations of a disease, which may be overlooked if one focuses too closely on only a certain set of molecular parameters for disease diagnosis.

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18

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Image of FIGURE 1
FIGURE 1

Qiagen Biorobot 9604 automated DNA extraction system. (Courtesy of Qiagen.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 2
FIGURE 2

Qiagen Biorobot 9604 Tip-Change System and bar-coded samples. (Courtesy of Qiagen.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 3
FIGURE 3

The ABI PRISM 6700 instrument combines extraction technology with preparation of PCR master mix. Specimens and reagents (rgt's) are mixed in plates, which are sealed and PCR ready. RSP, robotic sample preparation. (Courtesy of ABI.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 4
FIGURE 4

The Roche MagNA Pure LC uses magnetic-bead technology for nucleic acid extraction and is programmable to load extracted samples into glass LightCycler capillary tubes in their rotor housing. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 5
FIGURE 5

Fluorescent excitation and emission spectra of a sample donor and acceptor dye pair. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 6
FIGURE 6

Schematic of a molecular-beacon probe binding to denatured DNA template. As the beacon probe binds, the quencher molecule is removed from proximity to the fluorophore, allowing the dye to fluoresce.

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 7
FIGURE 7

Schematic of TaqMan chemistry. Hybridization probes, labeled with a reporter dye and a fluorescent quencher molecule, are bound to the denatured target DNA strand. As PCR occurs and the opposing strand is generated, the 5′-nuclease activity of DNA polymerase will hydrolyze the probe, removing the reporter from its close proximity to the quencher dye and allowing measurable fluorescent signal to accumulate. (Courtesy of ABI.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 8
FIGURE 8

ABI PRISM 7700 sequence detection system. (Courtesy of ABI.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 9
FIGURE 9

Amplification plots for the ABI PRISM 7700 sequence detection system. Negative controls are used to establish fluorescent-signal background thresholds. As exponential amplification of a specific target occurs, the amplification curve for a positive sample will cross those thresholds. The PCR cycle number at which a curve crosses the threshold is related to the original concentration of nucleic acid in the sample and can provide a measure of quantitation to the assay. (Courtesy of ABI.) ΔRn, change in relative fluorescence; exp, experiment.

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 10
FIGURE 10

LightCycler system for real-time PCR. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 11
FIGURE 11

Sealed glass capillaries house the PCR mix and the sample. The reaction tubes are placed in a circular rotor within the LightCycler system for rapid thermal cycling and amplicon detection. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 12
FIGURE 12

The LightCycler fluorimeter monitors fluorescent changes as they occur when DNA is amplified in glass reaction tubes seated in the circular rotor. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 13
FIGURE 13

FRET. A pair of oligonucleotide probes is designed to hybridize in close proximity to one another on the target DNA strand. As oligonucleotide probes, synthesized to contain a fluorescent dye, are bound to the target DNA strand, energy emitted from fluor 1 (F) will excite fluor 2 (F) to emit a specific wavelength that is monitored by the LightCycler instrument. Fluorescent signals provide a measure of real-time PCR and specific hybridization of probes. (Courtesy of Roche Molecular Biochemicals.) hυ, light.

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 14
FIGURE 14

Melting curve analysis for FRET-based probes. Fluorescence will diminish as the temperature is increased, since probes will be removed from the target DNA strands. Mismatches in the target sequence (mutations) will allow the probe to be removed more easily than the same probe bound to a target sequence that is an exact match for the probe (wild type). By plotting the ratio of fluorescent signal to temperature, the temperature at which most of the melting takes place can be monitored and wild-type sequences can be distinguished from those of mutants. (Courtesy of Roche Molecular Biochemicals.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 15
FIGURE 15

iCycler. (Courtesy of Bio-Rad.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 16
FIGURE 16

The Amplifluor Universal Amplification and Detection System uses traditional PCR primers, one of which synthesizes with a tail (the Z region). The Z region is replicated as the opposing strand is generated. Primers are combined with a fluorescent hairpin oligonucleotide, called a Uniprimer, that binds specifically to the Z regions. Fluorescent signal is generated when a Uniprimer unfolds during incorporation into an amplification product. (Courtesy of Intergen.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 17
FIGURE 17

Schematic of the Invader assay. Image created by Jodi Hoeser. (Courtesy of Third Wave Technologies, Inc.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 18
FIGURE 18

Schematic of a hybridization experiment using the GeneChip probe array. (Courtesy of Affymetrix.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 19
FIGURE 19

Close up of probe array surface. Various fluorescent- signal intensities, depicted by color hybridization displays, are generated and digitized. A stronger signal is produced when there is an exact match between the probe and the target. Therefore, the resulting signal is a measure of both sequence homology and the quantity of DNA bound to the chip. Semiquantitative hybridization information is obtained. (Courtesy of Affymetrix.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 20
FIGURE 20

The GeneChip instrument system with fluidics station, hybridization oven, Agilent GeneArray to measure emitted light, PC workstation, and GeneChip Data Analysis Suite software. (Courtesy of Affymetrix.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 21
FIGURE 21

Electropherogram produced by capillary electrophoresis. (Courtesy of Paul Rys, Mayo Clinic, Rochester, Minn.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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Image of FIGURE 22
FIGURE 22

Pyrosequencing chemistry. A sequencing primer is hybridized to a DNA template and incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, and the substrates adenosine 5′-phosphosulfate and luciferin. As complementary deoxynucleoside triphosphates are added individually to the reaction mixture, they are incorporated into short to medium-length DNA strands, and pyrophosphate is released in a quantity equal to that of the original incorporated nucleotides. ATP is produced and drives the luciferase reaction so that light is generated. (Courtesy of Pyrosequencing.)

Citation: Wolk D, Persing D. 2002. Clinical Microbiology: Looking Ahead, p 429-450. In Truant A (ed), Manual of Commercial Methods in Clinical Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555817961.ch18
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