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Chapter 3 : In Vitro Nucleic Acid Amplification Techniques
Category: Clinical Microbiology; Bacterial Pathogenesis
Nucleic acid amplification (NAA) techniques have come of age. The specific amplification and detection of an oligonucleotide sequence went from the fictional to the mundane in the span of two decades. By looking at the theoretical basis for each method, NAA techniques can be placed into one of two broad categories: (i) target amplification systems, including PCR, ligase chain reaction (LCR), self-sustaining sequence amplification (3SR), nucleic acid sequence-based amplification (NASBA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP); and (ii) signal amplification systems (including probe amplification methods), such as branched-DNA technologies (bDNA) and cleavage-based signal amplification (cycling probe technologies [CPT] and Invader assays). Proofreading may help maintain fidelity of replication, and its absence can result in a relatively high rate of nucleotide incorporation errors (misincorporation), most relevant when starting with low target numbers. Misincorporation can produce amplicon mismatched to detection probes and can also result in inefficient amplification (especially when longer genetic stretches are targeted), due to primer mismatch in subsequent amplification rounds. Successful ligation relies on contiguous positioning and correct base pairing of the 3' and 5' ends of oligonucleotide probes on a target DNA molecule. The reliability of sequence data has improved, and the taxonomic and cytogenetic characterization of microorganisms has advanced tremendously. These improvements give the tools to allow the rapid and increasingly routine development of molecular methods for the identification, quantification, and characterization of microorganisms.
Use of NAA in clinical microbiology.
Comparative material and kit costs of culture-based and NAA-based diagnostic assays for infectious disease.
Fact or fallacy? While at face value these assumptions seem to be self-evident facts, such is not always the case.
Scheme for PCR. Reprinted from reference 236 with permission.
Scheme for LCR. Reprinted from reference 236 with permission.
Scheme for SDA. Reprinted from reference 236 with permission.
Scheme for transcription-based amplification. Reprinted from reference 236 with permission.
Scheme for LAMP. Reprinted from reference 148 with permission.
Scheme for bDNA signal amplification. Reprinted from reference 236 with permission.
Scheme for the Invader® assay. Reprinted from reference 236 with permission.