CHAPTER 4. “DNA Techniques” Case Study: Isothermal Approaches

Abstract

Modern molecular diagnostics is built upon nucleic acid amplification (NAA) technologies, which have revolutionised the detection and quantitation of biological materials. Emerging isothermal NAA approaches offer a range of advantageous performance characteristics, such as reduced susceptibility to matrix interferents and minimal instrumentation requirements whilst providing a viable alternative to ‘gold standard’ polymerase chain reaction (PCR)-based methodologies. Loop-mediated isothermal amplification (LAMP) is currently the main focus for isothermal NAA method development activities within the research and commercial diagnostic communities. LAMP technology is well suited to diagnostic applications due to factors such as reaction speed and a tolerance to common matrix interferents. Newer isothermal NAA technologies such as cross priming amplification (CPA) and recombinase polymerase amplification (RPA) are also moving into the testing space with the potential for enhanced analytical performance. The food molecular diagnostics sector includes challenging areas, such as authenticity testing, that routinely support legislative frameworks. Isothermal NAA technologies are central to new diagnostic tools, supporting applications such as meat speciation and genetically modified organism (GMO) detection within laboratory and field settings. Isothermal NAA technologies will have an important and evolving role to play in the future of food molecular diagnostics by expanding availability and testing capabilities within the general diagnostic community, and resource-limited and point-of-use environments.