Molecular diagnostics refers to a broad range of techniques used to analyse biomarkers, such as DNA, RNA, and proteins to identify and monitor disease. The field evolved from key discoveries in molecular biology, beginning with the identification of DNA's double-helix structure in 1953. Significant progress was made by the 1980 with the development of rapid amplification techniques including Polymerase Chain Reaction (PCR) and isothermal amplification. The techniques made it possible to detect analytes in small quantities and by the 1990s, molecular diagnostics entered clinical practice, offering tools for detecting genetic diseases, pathogens, and cancer biomarkers.

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Advances in the 2000s, such as real-time PCR and Next-Generation Sequencing (NGS), aim to expand the application of molecular diagnostics while offering improved precision. However, much of the diagnostic process remains dependant on specialised (and often expensive) equipment, requiring samples to be sent to centralised laboratories for processing. In healthcare, this process can result in patients waiting days or weeks for a diagnosis, delaying treatment and potentially impacting patient outcomes. More recently, portable technologies, such as COVID-19 lateral flow tests, have begun to bring molecular diagnostics to the point-of-care, facilitating timely diagnosis.

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Common Techniques

Four major techniques form the cornerstones of modern molecular diagnostics[1]:

• Polymerase chain reaction (PCR) - Amplification

Considered the gold standard in molecular diagnostics, PCR uses thermal cycling to amplify target nucleic acid. This method enables the detection of very small amounts of specific DNA targets within small sample quantities with high specificity and sensitivity. Variants like real-time PCR (qPCR) allow for quantification, while reverse transcription PCR (RT-PCR) is used to analyse RNA.

• Isothermal amplification - Amplification

Similar to PCR, isothermal techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) amplify nucleic acid at constant temperatures.

• Next-generation sequencing (NGS) - Sequencing

While PCR and isothermal techniques amplify nucleic acid to enable detection, they do not sequence the resulting product. NGS techniques can be applied post amplification to confirm the presence of  expected amplicons or identify specific strains, subtypes, or mutations.

• Clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods - Sequencing

While known best as a gene editing tool, CRISPR is also emerging as a powerful diagnostic tool.