Abstract
Nucleic acid amplification technologies (NAATs) have become fundamental tools in molecular diagnostics, due to their ability to detect small amounts of target molecules. Since its development, Polymerase Chain Reaction (PCR) has been the most exploited method, being stablished as the “gold standard” technique for DNA amplification. However, the requirement for different working temperatures leads to the need of a thermocycler machine or complex thermal apparatus, which have been preventing its application in novel integrated devices for single workflow and high throughput analysis. Conversely, isothermal amplification methods have been gaining attention, especially for point-of-care diagnosis and applications. These non-PCR based methods have been developed by mimicking the in vivo amplification mechanisms, while performing the amplification with high sensitivity, selectivity and allowing for high-throughput analysis. These favorable capabilities have pushed forward the implementation and commercialization of several platforms that exploit isothermal amplification methods, mostly against virus, bacteria and other pathogens in water, food, environmental and clinical samples. Nevertheless, the future of isothermal amplification methods is still dependent on achieving technical maturity and broader commercialization of enzymes and reagents.
Highlights
Innovations in biotechnology and molecular biology have push forward novel nucleic acid amplification technologies (NAATs) to meet the demands for more efficient, sensitive, specific protocols capable to be integrated into portable systems and for high-throughput analyses
This small but critical bottleneck has spurred the development of non-Polymerase Chain Reaction (PCR) based techniques, preferably suitable to be performed at a constant temperature—isothermal amplification (IA) methods, which aim at simplifying protocols for point-of-care (PoC) use, allowing to move the molecular diagnosis from centralized labs
Recombinase Polymerase Amplification (RPA) covers for circa 20%, and the remainders, RPA, Rolling Circle Amplification (RCA), Helicase Dependent Amplification (HDA) and Nucleic Acid Sequence-based Amplification (NASBA) add up to 5%
Summary
Innovations in biotechnology and molecular biology have push forward novel nucleic acid amplification technologies (NAATs) to meet the demands for more efficient, sensitive, specific protocols capable to be integrated into portable systems and for high-throughput analyses. Inherent features to PCR require dedicated instrumentation capable of thermal cycling with appropriate temperature control, which is usually found in laboratory settings This small but critical bottleneck has spurred the development of non-PCR based techniques, preferably suitable to be performed at a constant temperature—isothermal amplification (IA) methods, which aim at simplifying protocols for point-of-care (PoC) use, allowing to move the molecular diagnosis from centralized labs. While PCR requires the lowering and rising of two or three working temperatures, to allow annealing and denaturation of DNA strands and primers, and to facilitate strand displacement, IA methods proceed at a single working temperature, which removes the need for a thermocycler Some of these isothermal amplification mechanisms allow direct amplification from non-DNA targets, such as RNA (without the need for an additional retro transcription step) or protein-nucleic acids conjugates. Scientific literature has been reporting the application of isothermal amplification systems in a wide number of fields, among them: pathogen detection in clinical, environmental and food samples (Fang et al, 2010; Chow et al, 2008; Zhao et al, 2012; Mahmoudian et al, 2008a; Krõlov et al, 2014), diagnosis of various infectious diseases
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