Isothermal Amplification of Nucleic Acids: The Race for the Next “Gold Standard”

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.

Loop-Mediated Isothermal Amplification for Detection of Plant Pathogens in Wheat (Triticum aestivum)

“…mLAMP assays applied to detect numerous species also need to be designed carefully to avoid interference or non-desired interactions between primers. In contrast, microfluidic technology for parallelized LAMP allows for detection of up to 1,200 samples simultaneously while maintaining sensitivity ( Oliveira et al, 2021 ). Some mLAMP techniques have shown great potential to be applied for detection of wheat pathogens ( Yasuhara-Bell et al, 2018 ; Kang et al, 2020 ), while parallelized LAMP using microfluidics is starting to become popular for plant pathogen detection with some examples involving plant viruses ( Natsuhara et al, 2020 ).…”

“…Additionally, the use of only two primers in RPA reactions compared to six for LAMP simplifies primer design, and use of the recombinase polymerase lowers detection time. However, the lower reaction temperatures (between 30°C and 55°C) make RPA more prone to non-specific primer binding compared to other isothermal amplification techniques, which can cause amplification of non-target templates ( Oliveira et al, 2021 ). Other limitations of RPA include amplification of only small DNA fragments of less than 1,500 bp with long primers (30–50 nt), which can yield non-specific amplification and a highly variable sensitivity ( Ivanov et al, 2021 ).…”

Short prokaryotic Argonaute system repurposed as a nucleic acid detection tool

“…Accessible, rapid, and sensitive tools that can be used on the field, at the point‐of‐care, or at home, will greatly benefit diagnostic purposes. The need for thermocyclers required for PCR has been circumvented by the development of isothermal amplification techniques, during which the target sequence is amplified at a constant temperature 4 . Akin to PCR, also this technique can be combined with fluorescence and colourimetric detection methods 4 .…”

“…The need for thermocyclers required for PCR has been circumvented by the development of isothermal amplification techniques, during which the target sequence is amplified at a constant temperature 4 . Akin to PCR, also this technique can be combined with fluorescence and colourimetric detection methods 4 . However, many isothermal amplification techniques face challenges including high background signal, complex primer design, and false‐positives 4 which slowed down their broader commercialization.…”

Label-free electrical monitoring of nucleic acid amplification with integrated hydrogel ionic diodes

“…To date, a wealth of nucleic acid amplification techniques have been broadly applied, such as polymerase chain reaction (PCR) [ 6 ], recombinase polymerase amplification (RPA) [ 7 ], and multiple displacement amplification (MDA) [ 8 ]. Monitoring the progress of such assays is critical to determining the existence and levels of target nucleic acids, which typically incorporates fluorescent reporter probes and optical quantification means [ 9 , 10 ]. However, the fluorescent reagents may pose inhibitory effects to the reactions [ 11 ].…”