Chapter 15 : Quantitative Molecular Techniques

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This chapter describes the quantitative molecular techniques that serve as the basis of viral load assays, and discusses key issues and important variables that affect assay performance. Real-time methods offer the advantages of increased dynamic range, simplicity, reduced analysis time, and diminished risk of contamination. Although polymerase chain reaction (PCR) is the best developed and most widely used nucleic acid amplification strategy, other strategies have been developed and several serve as the basis of quantitative assays for viral nucleic acids. Quantitative assays based on nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA), and hybrid capture are commercially available. The specificity of real-time PCR can also be increased by including hybridization probes in the reaction mixture. NASBA is one of several transcription-based amplification methods that amplify RNA targets. Hybrid capture assays (HCAs) employ a signal amplification technology that can be applied to the detection and quantitation of DNA or RNA target molecules without amplification. Prior to the development of quantitative molecular techniques, laboratories were limited to cumbersome culture methods or insensitive antigen detection methods to determine viral load. Each of the quantitative molecular techniques has particular strengths and limitations that are inherent in the underlying nucleic acid amplification strategies. A thorough understanding of the key performance issues and features of the available quantitative molecular techniques is essential to the practice of modern clinical virology.

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 1

Schematic of the Roche Amplicor Monitor system for the quantitation of HIV-1 RNA. QS, quantitation standard; OD, optical density. (Provided by Roche Molecular Systems.)

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 2

Real-time PCR amplification plot with commonly used terms and abbreviations. Rn , normalized fluorescent signal from reporter dye.

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 3

Example of a standard curve for a real-time PCR assay with the regression line equation and R 2 statistic. The efficiency of the PCR amplification can be estimated using the following equation: efficiency = 10–slope – 1.

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 4

NASBA pathways for single-stranded RNA (A) and double-stranded DNA (B). RT, avian myeloblastosis virus RT; pol, T7 RNA polymerase; wavy lines, RNA; dashed lines, newly synthesized DNA; solid lines, primers. (Provided by Organon-Teknika.)

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 5

Principle of viral nucleic acid quantitation with NASBA. WT, wild-type sequence; Q, calibrator; ECL, electrochemiluminescence. (Provided by Organon-Teknika.)

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 6

Diagram of a third-generation bDNA assay. (Provided by Siemens.)

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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Image of FIGURE 7

Diagram of an HCA. The specimen is denatured (1) and then hybridized with an RNA probe (2). If the target nucleic acid is present, the resulting DNA-RNA hybrid is captured by an antibody specific for the hybrids (3). Multiple alkaline-phosphatase-conjugated antibodies bind to the captured hybrids (4). A chemiluminescent substrate emits light that is measured in a luminometer (5). (Provided by Digene.)

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15
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FDA-cleared viral load assays a

Citation: Nolte F. 2009. Quantitative Molecular Techniques, p 169-184. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch15

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