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Chapter 6 : Molecular Microbiology*

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Molecular Microbiology*, Page 1 of 2

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Abstract:

Nucleic acid amplification techniques are now commonly used to diagnose and manage patients with infectious diseases. The growth in the number of FDA-cleared test kits and analyte-specific reagents has facilitated the use of this technology in the clinical laboratory. Technological advances in nucleic acid amplification techniques, automation, nucleic acid sequencing, and multiplex analysis have revitalized the field of molecular microbiology and created new opportunities for growth. Simple, sample-in, answer-out molecular test systems are now available that can be deployed in a variety of laboratory and clinical settings. Molecular microbiology remains the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. Nucleic acid-based tests have reduced the dependency of the clinical microbiology laboratory on more traditional antigen detection and culture-based methods and created new opportunities for the laboratory to affect patient care. This chapter covers nucleic acid probes, signal and target amplification techniques, postamplification detection and analysis (e.g., electrophoresis, hybridization, sequencing, microarrays, and mass spectrometry), clinical applications of these techniques, and the special challenges and opportunities that these techniques provide for the clinical laboratory.

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 1
FIGURE 1

PNA probes for (green), (yellow), and (red). Reprinted with permission of AdvanDx from http://www.advandx.com/Technology/image-gallery. doi:10.1128/9781555817381.ch6.f1

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 2
FIGURE 2

bDNA signal amplification. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f2

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 3
FIGURE 3

Hybrid capture signal amplification. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f3

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 4
FIGURE 4

Cleavase-invader probe-based amplification. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f4

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 5
FIGURE 5

PCR target amplification. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f5

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 6
FIGURE 6

Real-time PCR amplification plot with commonly used terms and abbreviations. C, cycle threshold; R, normalized fluorescent signal from reporter dye. From , p 5-4 (Applied Biosystems, Foster City, CA, 2010). Reprinted with permission. doi:10.1128/9781555817381.ch6.f6

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 7
FIGURE 7

5′ Exonuclease chemistry for real-time PCR applications. Modified with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f7

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 8
FIGURE 8

Dual hybridization probes for real-time PCR applications. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f8

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 9
FIGURE 9

Molecular beacon probes for real-time amplification applications. Modified with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f9

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 10
FIGURE 10

Scorpion probes. (A) The primer element binds to the DNA target. The probe element is in the closed, nonfluorescent configuration. (B) The primer is extended and incorporates a probe-binding site into the new strand. The probe element remains in the closed, nonfluorescent configuration. (C) After a cycle of denaturation and reannealing, the probe flips forward to bind its target site on the same molecule. The fluorophore and quencher are now separated, and the fluorescence increases. Reprinted with permission from . doi:10.1128/9781555817381.ch6.f10

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 11
FIGURE 11

Principles of partially double-stranded linear probes. A double-stranded linear probe contains two complementary oligonucleotides of very different lengths. The longer, positive strand is 5′ labeled with a fluorophore reporter, while the shorter, negative strand carries a quencher moiety at its 3′ end. When not bound to a target, the probe is nonfluorescent due to the close proximity of the fluorophore and the quencher. In the presence of a specific target, the positive strand preferentially hybridizes to the target, resulting in increased fluorescence signal generation. Modified from . doi:10.1128/9781555817381.ch6.f11

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 12
FIGURE 12

Digital PCR works by partitioning a sample into many nanoliter-scale individual PCR reactions, some of which contain a target molecule (red) while others do not (gray). Following PCR, the ratio of positive to negative reactions is used to calculate the starting number of target molecules. gDNA, genomic DNA. Reprinted with permission from Applied Biosystems QuantStudio OpenArray Digital PCR Application Note. doi:10.1128/9781555817381.ch6.f12

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 13
FIGURE 13

Transcription-based target amplification. NASBA and TMA are examples of transcription-based amplification systems. Reprinted with permission of Elsevier from . doi:10.1128/9781555817381.ch6.f13

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 14
FIGURE 14

Strand displacement target amplification. Modified from . doi:10.1128/9781555817381.ch6.f14

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 15
FIGURE 15

(a) Primer design of the LAMP reaction. For ease of explanation, six distinct regions are designated on the target DNA, labeled F3, F2, F1, B1c, B2c, and B3 from the 5′ end. As c represents a complementary sequence, the F1c sequence is complementary to the F1 sequence. Two inner primers (FIP and BIP) and outer primers (F3 and B3) are used in the LAMP method. FIP (BIP) is a hybrid primer consisting of the F1c (B1c) sequence and the F2 (B2) sequence. (b) Starting structure-producing step. DNA synthesis initiated from FIP proceeds as follows. The F2 region anneals to the F2c region on the target DNA and initiates the elongation. DNA amplification proceeds with BIP in a similar manner. The F3 primer anneals to the F3c region on the target DNA, and strand displacement DNA synthesis takes place. The DNA strand elongated from FIP is replaced and released. The released single strand forms a loop structure at its 3′ end (structure 3). DNA synthesis proceeds with the ssDNA as the template and BIP and B3 primer, in the same manner as described earlier, to generate structure 5, which possesses the loop structure at both ends (dumbbell-like structure). (c) Cycling amplification step. Using self-structure as the template, self-primed DNA synthesis is initiated from the 3′-end F1 region, and the elongation starts from FIP, annealing to the single strand of the F2c region in the loop structure. Passing through several steps, structure 7 is generated, which is complementary to structure 5, and structure 5 is produced from structure 8 in a reaction similar to that which led from structures 5 to 7. Structures 9 and 10 are produced from structures 6 and 8, respectively, and more elongated structures ( ) are also produced. Reprinted with permission of Nature Publishing Group from . doi:10.1128/9781555817381.ch6.f15

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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Image of FIGURE 16
FIGURE 16

HDA amplifies target sequences using two sequence-specific primers flanking the fragment to be amplified and a mixture of enzymes for DNA strand separation and polymerization. In the first step of the HDA reaction, the helicase enzyme loads onto the template and traverses along the target DNA, disrupting the hydrogen bonds linking the two strands. Exposure of the single-stranded target region by helicase allows primers to anneal. The DNA polymerase then extends the 3′ ends of each primer using free deoxynucleotides (dNTPs) to produce two DNA replicates. The two replicated DNAs independently enter the next cycle of HDA, resulting in exponential amplification of the target sequence. Reprinted with permission of BioHelix from http://www.biohelix.com/HDA_mechanism.asp. doi:10.1128/9781555817381.ch6.f16

Citation: Nolte F. 2015. Molecular Microbiology*, p 54-90. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch6
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