Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout
- Lauren M. Aufdembrink1,
- Pavana Khan1,
- Nathaniel J. Gaut2,
- Katarzyna P. Adamala1,2 and
- Aaron E. Engelhart1,2
- 1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA
- 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA
- Corresponding authors: enge0213{at}umn.edu, kadamala{at}umn.edu
Abstract
Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.075192.120.
- Received February 25, 2020.
- Accepted May 26, 2020.
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