Chapter 4 : Molecular Microbiology

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Technological advances in realtime PCR techniques, automation, nucleic acid sequencing, multiplex analysis, and mass spectrometry have reinvigorated the field and created new opportunities for growth. Molecular microbiology is the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. This chapter covers amplified- and nonamplified-probe techniques, postamplification detection and analysis, clinical applications of these techniques. It also discusses the special challenges and opportunities that these techniques provide for the clinical laboratory. Although probes can range from 15 to thousands of nucleotides in size, synthetic oligonucleotides of less than 50 nucleotides are commonly incorporated into commercial kits. In most clinical applications, formalin-fixed, paraffin-embedded tissue sections are used. Nonamplified-probe techniques are used most effectively in culture confirmation assays for mycobacteria and systemic dimorphic fungi. Strategies other than PCR, are based on signal, target, or probe amplification, and have sensitivity unparalleled in laboratory medicine, have created new opportunities for the clinical laboratory to have an effect on patient care, and have become the new “gold standards” for laboratory diagnosis of many infectious diseases. In signal amplification methods, the concentration of the probe or target does not increase. Signal amplification assays have several advantages over target amplification assays. The target amplification systems share certain fundamental characteristics. They use enzyme-mediated processes, in which a single enzyme or multiple enzymes synthesize copies of target nucleic acid. In these techniques, the amplification products are detected by two oligonucleotide primers that bind to complementary sequences on opposite strands of double-stranded targets.

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 1

bDNA signal amplification. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 2

PCR target amplification. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 3

Real-time PCR amplification plot with commonly used terms and abbreviations. R, normalized fluorescent signal from reporter dye. From , p. 5–94 (Applied Biosystems, Foster City, CA, 2002). Reprinted with permission.

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 4

5′ exonuclease chemistry for real-time PCR applications. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 5

Dual hybridization probes for real-time PCR applications. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 6

Molecular beacon probes for real-time amplification applications. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 7

Transcription-based target amplification. NASBA and TMA are examples of transcription- based amplification systems. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 8

Strand displacement target amplification. The process is shown for only one strand of a dsDNA target, but amplification occurs on both strands simultaneously. B and B, bumper primers; S and S, amplification primers. Modified from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Image of FIGURE 9

Cleavase-invader probe-based amplification. Reprinted with permission of Elsevier from reference .

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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Comparison of next-generation and Sanger sequencing platforms

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4
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FDA-cleared or -approved molecular diagnostic tests for infectious diseases as of 10 December 2009

Modified from http://www.amp.org. Abbreviations: ASPE, allele-specific primer extension; HC, hybrid capture; HPA, hybridization protection assay; HR, high risk; JBAIDS, Joint Biological Agent Identification and Diagnostic System; PNA FISH, peptide nucleic acid fluorescent hybridization; SSTI, skin and soft tissue infection; TC, target capture.

Five species directly from positive blood cultures, including and/or , , and and/or .

Mycobacteria include , , complex, , , and complex. Fungi include , , , and . Bacteria include spp., spp., group , group , , , , , and .

Viruses include influenza A (H1, H3) virus; influenza B virus; adenovirus; respiratory syncytial viruses A and B; metapneumovirus; parainfluenza virus types 1, 2, and 3; and rhinovirus.

Viruses include influenza A and B viruses and respiratory syncytial virus.

Viruses include influenza A and B viruses and respiratory syncytial virus.

Citation: Nolte F, Caliendo A. 2011. Molecular Microbiology, p 27-59. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch4

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