Molecular Diagnostics

Abstract

Molecular diagnostics in infection generally relate to the detection and/ or characterization of nucleic acid sequences of infectious agents in clinical samples which are used to provide: ● A laboratory diagnosis. ● A means of monitoring patients at risk of developing disease caused by a particular infection. ● A method to predict through genotypic analysis the susceptibility or resistance to appropriate treatments. ● A measurement of the response to therapy. A few key laboratory techniques underpin the majority of molecular diagnostic tests that are currently used in the field of infection, and include: ● Block-based polymerase chain reaction (PCR). ● Real-time PCR, including quantification. ● Strand displacement amplification. ● Transcription mediated amplification. ● DNA sequencing. These can be commercially sourced, which has the advantage of CE marking, or developed in-house, sometimes referred to as laboratory developed tests (LDTs). Whatever the source, the underlying principles are often the same and rigorous evaluation and validation is required for the adoption of any molecular test in the diagnostic laboratory. The majority of molecular diagnostic tests require the amplification of a specific DNA sequence and its subsequent detection by a variety of means. As such, small sequences of DNA from the infectious agent are amplified from a relatively low copy number in the clinical sample. For example, after thirty to forty cycles of PCR, a single copy of a sequence can theoretically be amplified to over a billion copies. This PCR product, commonly termed amplicon, can provide a template for any further testing with the same PCR test and therefore potentially act as a source for false positive results. Molecular diagnostic laboratories have requirements to keep the different stages of the molecular test separate and minimize the risk of amplicon contamination. Most facilities will have a ‘clean PCR laboratory’ that is used to store the clean reagents such as primers, probes, enzyme mastermixes, and no clinical samples, nucleic extracts, or amplification reactions are ever taken into this environment. Another laboratory is used for the nucleic acid extraction of the clinical samples and this environment is often used to set up the PCR reactions.