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Chapter 5 : Quantitative Molecular Methods
Category: Clinical Microbiology; Bacterial Pathogenesis
Quantitative molecular methods provide information about the concentration of microbial nucleic acid target present in a sample. The evolution of various quantitative methods includes PCR and commercial alternatives to PCR. Quantitative methods that rely on target amplification include transcription-mediated amplification (TMA) and nucleic acid sequenced-based amplification (NASBA). Commercially available quantitative signal and probe amplification methods include the branched DNA (bDNA) method and the Invader assay, respectively. Quantitative methods based on PCR include three broad categories: quantitative PCR (Q-PCR) is typically used to determine the microbial density or ‘‘load’’ of DNA in clinical specimens; (ii) quantitative reverse transcriptase PCR (QRT-PCR) is used to determine the density of RNA viruses, an approach commonly called ‘‘viral load’’ testing; and (iii) in what are often referred to as ‘‘gene expression’’ assays, QRT-PCR can be used to determine relative mRNA expression levels for different disease states. This chapter concentrates on the technological features unique to quantitative molecular assays. The advantages and limitations of these methods are reviewed in the chapter. For microbial quantitation, whole-organism comparisons are best but not always feasible; therefore, plasmids or oligonucleotides are often used as substitutes. The chapter talks about general issues for viral load measurement, and other general considerations and tips for quantitative methods. Speed, accuracy, and utilization of results will be paramount to the future of quantitative technology as new methods extend our understanding of pathogenesis and advance our ability to improve diagnosis and disease management.
Plot of the relative amount of DNA product as a function of amplification efficiency in a 25-cycle PCR assay. The relative final quantity at each amplification efficiency is a percentage of the final amount of PCR product amplified at maximal efficiency (1.0). The inset shows expansion of the 0.7-to-0.9 range.
(A) Fluorescence output from real-time PCR showing eight samples with corresponding copy numbers and corresponding standard curves derived from the C T values. Reactions occurring after cycle 45 could depict the formation and amplification of primer-dimers, so sequence analysis of these types of reaction may be necessary. (B) Example of a standard curve derived from real-time Q-PCR with calculations for PCR efficiency. The log of the starting copy number of nucleic acid is plotted against C T . Samples of unknown concentration can be assessed in comparison to the standard curve. Efficiency can be calculated from the slope of the regression line.
Diagram of internal control sequences for PCR-based assays. (A) Native template showing the internal probe binding site in the normal orientation. (B and C) Control sequences identical to sequence A but with either deletion of an internal sequence (B) or insertion of an exogenous sequence (C). (D) Control sequence, the same size as the native sequence, containing a substituted sequence. (E) Control sequence with an internal probe-binding sequence in reverse orientation.