Methods for digital readout quantification of nucleic acids

Abstract

The invention provides methods for detecting specific nucleic acids using a loop-mediated isothermal amplification (LAMP) reaction and providing a digital readout of the results. One method teaches separating target specificity from indicator detection in a two-stage multiple-threshold array (MTA). During the first stage amplicons incorporating both target sequence and an indicator sequence are synthesized. During the second stage of the reaction, the indicator sequence is targeted and amplified to produce visual results that may be digitalized. Another method teaches a competitive MTA, in which a reference sequence is used to compete against target sequence during amplification producing threshold responses, thus enabling digital readout. Using either method, quantitation is achieved without the need for continuous monitoring of the reaction and the end-point readout is amenable to visual inspection. Methods of the invention are especially useful in circumstances where there is lower or intermittent power supply.