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Chapter 14 : Nucleic Acid Amplification and Detection Methods

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Abstract:

Nucleic acid detection methods play an increasingly important role in the detection of viral infection. This chapter describes the major nucleic acid testing methods and assists in test selection. Nucleic acid amplification methods are classified as target or probe amplification methods based upon the source of the nucleic acid that is amplified in the procedure. Target amplification methods are among the oldest and best characterized nucleic acid amplification methodologies. Nucleic acid amplification detection methods include polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA). Nucleic acid detection methods include SYBR green, fluorescence resonance energy transfer (FRET) system and hydrolysis (TaqMan) probes. Other amplification methods described in the chapter are part of closed assay systems whose manufacturers discourage or prohibit in-house development. Nucleic acid detection methods have also made significant improvements in one's ability to detect fastidious and slow-growing viruses (e.g., human parvovirus, Epstein-Barr virus, and certain enteroviruses), viruses that are dangerous to amplify in culture (e.g., human immunodeficiency virus and certain hemorrhagic fever viruses), and viruses that are present in low concentrations. While nucleic acid detection methods will never completely replace culture and direct fluorescent antibody methods, nucleic acid detection methods will continue to play an important role in the detection and monitoring of viral diseases.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Figures

Image of FIGURE 1
FIGURE 1

PCR. The dsDNA target (top) is heated to separate the strands. As the solution cools, the two oligonucleotide primers bind to opposite strands on the target DNA. The thermostable Taq DNA polymerase extends the primers according to the nucleotide sequence of the target DNA strand to produce dsDNA products. The old and new strands serve as templates for further DNA synthesis during the next cycle of heating and cooling. dNTPs, deoxynucleoside triphosphates.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 2
FIGURE 2

NASBA and TMA. In these procedures, primer A, containing the promoter sequence for the T7 RNA polymerase and a sequence complementary to the target RNA, binds to the target RNA strand (top). RT extends the primer according to the genetic sequence of the target strand, and RNase H degrades the RNA portion of the DNA-RNA hybrid molecule. Primer B binds to the complementary DNA, and RT extends the primer to make a complete, transcription-competent dsDNA intermediate. The T7 RNA polymerase generates 50 to 1,000 antisense (from the original RNA) RNA transcripts, each of which can be converted to transcription-competent dsDNA as before.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 3
FIGURE 3

SDA. An oligonucleotide primer containing a BsoB1 restriction site (5′-CTCGGG) binds to the complementary target nucleic acid. The primer and target are extended by a thermo-stable, exonuclease-deficient (exo) Bst DNA polymerase in the presence of dGTP, dATP, dUTP, and a dCTP that contains an alphathiol group (dCTPαS). The resulting DNA synthesis generates a double-stranded BsoB1 recognition site, with one strand containing 5′ phosphorothiolate linkages (shown as asterisks). BsoB1 nicks the strand without cutting the complementary thiolated strand, and the exo Bst polymerase extends the nucleic acid strand from the nick. The orignal nucleic acid is displaced rather than degraded because the DNA polymerase does not have 5′ exonucleolytic activity. The restriction site is regenerated by the polymerase. This linear amplification scheme becomes exponential when an antisense primer containing a BsoB1 site is added to the reaction mixture.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 4
FIGURE 4

Cleavase Invader assay. The invader probe and the signal probe hybridize adjacent to each other so that a portion of the signal probe does not hybridize to the target. The flap endonuclease (Cleavase) excises the unhybridized portion of the signal probe. The signal probe dissociates because the reaction is performed at or near the melting temperature for this probe. Another signal probe hybridizes, and the cycle repeats. The cleaved portion of the signal probe serves as an invader probe in the FRET cassette, and hybridization with the cassette produces an overlapping structure. The Cleavase enzyme excises the overlapping structure and separates the fluorophore (F) from the quencher (Q). The unquenched fluorophores accumulate in the reaction vessel.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 5
FIGURE 5

Typical EIA detection system. The target nucleic acids are heated and allowed to hybridize with biotinylated capture probes. The resulting mixture is placed into a microtiter plate well containing immobilized streptavidin (dark rectangles). The unhybridized nucleic acids are washed away, and a labeled detection probe is allowed to hybridize. After another wash, an appropriate substrate is added. A signal (color, fluorescence, or light, etc.) is generated when the target nucleic acid is present in the sample.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 6
FIGURE 6

Hybrid capture. The target DNA is denatured and allowed to hybridize to a large unlabeled RNA probe. The DNA-RNA hybrids are captured in a microtiter plate well by immobilized, hybrid-specific antibodies. Unbound materials are removed with a wash step. Labeled monoclonal antibodies to the DNA-RNA hybrid are added, and they bind to the entire length of the hybrid. After another wash, the chemiluminescent substrate is added. Light is generated if the target DNA was present in the sample.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 7
FIGURE 7

HPA. The oligonucleotide detector probe contains a chemiluminescent acridinium ester that is covalently attached to the probe via an acid-sensitive ether bond (top right). Complementary base pairing of the probe to the target protects the ether bond from acid hydrolysis. In the absence of base pairing, the ether bond is hydrolyzed and the label becomes permanently nonluminescent.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 8
FIGURE 8

Hydrolysis (TaqMan) probes. This method utilizes a reporter oligonucleotide that has a fluorescent dye (F) covalently coupled to the 5′ end and a quencher dye (Q) on the 3′ end. For PCR procedures, the reporter probe hybridizes internally to the flanking PCR primers. As the upstream primer is extended, the reporter oligonucleotide is displaced, then digested by the 5′→3′ nuclease activity of the polymerase (black oval). Digestion separates the reporter and quencher molecules and allows the reporter molecule to fluoresce strongly. Fluorescent reporter molecules accumulate with each amplification cycle.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 9
FIGURE 9

Hybridization probes. The hybridization probe format uses two oligonucleotides. The donor oligonucleotide is labeled at the 3′ end with a fluorescent dye (F), and the acceptor oligonucleotide (Q) is labeled at the 5′ end with a dye whose excitation frequency overlaps with the emission frequency of the donor dye. The probes are designed so that they hybridize in a head-to-tail arrangement on the target nucleic acid. If the target is present, the donor and acceptor dyes are in close proximity. FRET occurs when donor dye is excited by the light source. Light emitted by the donor dye then excites the acceptor dye, and the longer wavelength light emitted by the acceptor dye is detected by the instrument.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Image of FIGURE 10
FIGURE 10

Molecular beacon. Molecular beacons are stem-loop structures where the ends of the oligonucleotide are self-complementary and the center portion of the molecule is complementary to the target sequence (A). Self-annealing of the ends brings the fluorophore (F) and quencher (Q) dyes into close proximity, and the molecule will not fluoresce. In the presence of the target DNA (B), hybridization of the loop sequence is favored and the fluorophore and the quencher molecules are separated. The molecular beacon will fluoresce strongly in the presence of the target DNA.

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14
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Tables

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TABLE 1

General summary of nucleic acid amplification testing methods

Citation: Wiedbrauk D. 2009. Nucleic Acid Amplification and Detection Methods, p 156-168. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch14

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