Isothermal amplifications – a comprehensive review on current methods

Abstract

The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole.

A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens

“…HDA reacts with helicase, two primers, and two accessory proteins; methyl-directed mismatch repair (MutL), and single-stranded DNA (ssDNA) binding (SSB) proteins that stimulate UvrD helicase activity above ten-fold [37,38]. During the HDA replication process, the helicase unwinds double-stranded DNA (dsDNA) for the denaturation.…”

Framework Nucleic Acid-Based Spatial-Confinement Amplifier for miRNA Imaging in Living Cells

“…Developing methods for in situ sensitive monitoring of miRNAs within living cells first encounters challenges from the characteristics of miRNAs including sequence similarity and low abundance in total RNA samples . Isothermal enzymatic signal amplification-based fluorescence in situ hybridization (FISH) strategies that devoted to amplifying starting miRNA targets can be used to visualize gene expression at single-cell resolution. − Their applications in living cell imaging, however, are significantly challenged by the required cellular delivery of exogenous enzymes or other proteins, as well as the inevitable cell fixation, which may result in false signals and even the damage of cells. Nonenzymatic signal amplification methods, especially DNA amplifiers such as hybridization chain reaction (HCR) − and catalytic hairpin assembly (CHA), − are very promising for efficient detection of miRNA in cells due to their nonenzymatic and isothermal features.…”

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

“…Compared with PCR methods, isothermal amplification has the advantages of short reaction time and no need for qualified personnel or sophisticated equipment, and is a suitable candidate for POC application ( Glökler et al, 2021 ; Li and Macdonald, 2015 ). The common isothermal methods include loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) ( Notomi et al, 2000 ; Piepenburg et al, 2006 ).…”

“…Despite great progress, these isothermal amplification diagnostics often suffer from problems associated with nonspecific DNA amplification, followed by high rates of false-positive diagnosis ( Chen et al, 2021b ; Feng et al, 2021 ; Reid et al, 2018 ; Schneider et al, 2019 ; Tomita et al, 2008 ). Moreover, they are much less sensitive than RT-qPCR ( Glökler et al, 2021 ). In addition, although these isothermal methods can avoid complicated thermal cycling, some of them still require complex readout instruments that are challenging for personal use ( De Felice et al, 2022 ).…”