Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification

5 years ago

Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification

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

It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of “false” and “true” amplicons leads to order of magnitude quantitation by a single endpoint 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 by 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 without needing sophisticated material or informatics infrastructure. Lighting the LAMP: We developed loop-mediated isothermal amplification (LAMP) reactions transduced by oligonucleotide strand displacement (OSD) probes in which competition between defined numbers of “false” (introduced) and “true” (analyte) amplicons leads to order of magnitude analyte quantitation by a single visual endpoint determination. These LAMP-OSD reactions directly and quantitatively estimated numbers of nucleic acids in complex biospecimens.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700317