Multiplexed isothermal amplification and detection of nucleic acid sequences and biomarkers is of increasing importance in diverse areas including advanced diagnostics, food quality control and environmental monitoring. Whilst there are several very elegant isothermal amplification approaches, multiplexed amplification remains a challenge, requiring careful experimental design and optimisation, from judicious primer design in order to avoid the formation of primer dimers and non-specific amplification, applied temperature as well as the ratio and concentration of primers. In this review, we describe the various approaches that have been reported to date for multiplexed isothermal amplification, for both "one-pot" multiplexing as well as parallelised multiplexing using loop-mediated isothermal amplification, strand-displacement amplification, helicase-dependent amplification, rolling circle amplification, nucleic acid sequence-based amplification, with a particular focus on recombinase polymerase amplification.
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