Amplicon competition enables end-point quantitation of nucleic acids following isothermal amplification

Jiang, Yu Sherry; Stacy, Apollo; Whiteley, Marvin; Ellington, Andrew D.; Bhadra, Sanchita

It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We have now developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of ‘false’ and ‘true’ amplicons leads to order of magnitude quantitation via a single end-point determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout via cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics determinations without any sophisticated material or informatics infrastructure.