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Amplicon Competition Enables End‐Point Quantitation of Nucleic Acids Following Isothermal Amplification
Author(s) -
Jiang Yu Sherry,
Stacy Apollo,
Whiteley Marvin,
Ellington Andrew D.,
Bhadra Sanchita
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700317
Subject(s) - loop mediated isothermal amplification , amplicon , nucleic acid , isothermal process , chemistry , chromatography , biological system , polymerase chain reaction , computational biology , dna , biology , biochemistry , physics , thermodynamics , gene
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.