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.
Bibliographical noteFunding Information:
We thank Peter Unrau and members of the Engelhart laboratory for helpful discussions. This work was supported by NASA Contract 80NSSC18K1139 under the Center for Origin of Life (to A.E.E. and K.P.A.).
- Fluorescent aptamer
- Isothermal amplification
- Pathogen detection
- T7 RNA polymerase
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't