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

• Published in: Chembiochem : a European journal of chemical biology 2017-09, Vol.18 (17), p.1692-1695
• Main Authors: Jiang, Yu Sherry, Stacy, Apollo, Whiteley, Marvin, Ellington, Andrew D., Bhadra, Sanchita
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - genetics; Communication; Communications; Competition; copy number; Fusobacterium Infections - diagnosis; Fusobacterium nucleatum - genetics; Informatics; isothermal amplification; Mice; Neuropilin-2 - genetics; Neuropilin-2 - metabolism; Nucleic Acid Amplification Techniques; Nucleic acids; Nucleic Acids - genetics; Nucleic Acids - metabolism; point-of-care diagnostics; Point-of-Care Systems; Proto-Oncogene Proteins B-raf - genetics; Proto-Oncogene Proteins B-raf - metabolism; Quantitation; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sewage; signal threshold
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201700317

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.