Single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone is more robust than kinetic based real-time quantification

Abstract

This paper shows, using purified HIV RNA, that nucleic acid quantification in a digital format with reverse-transcription loop-mediated isothermal amplification (dRT-LAMP) is more robust than the standard real-time assay. Specifically, we show that the quantitative outcome of a dRT-LAMP reaction is insensitive to temperature changes over a 6 °C range, and that dRT-LAMP reactions can be imaged using a cell phone, with the resultant images being suitable for automatic analysis and quantification. We have previously shown success in quantification of HIV RNA with dRT-LAMP [1], with that study focused specifically on improving reaction efficiency. We have also previously shown, using the digital recombinase polymerase amplification, that digital reactions can be temperature tolerant [2]; here we extend this previous work by directly comparing digital vs. real time performance, as well as adding further experimental perturbations of non-quantitative imaging and automatic analysis.

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