Poster abstracts
Poster number 54 submitted by Pavana Khan
Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout
Lauren M. Aufdembrink (Genetics, Cell Biology and Development, University of Minnesota), Pavana Khan (Genetics, Cell Biology and Development, University of Minnesota), Nathaniel J Gaut (Genetics, Cell Biology and Development, University of Minnesota), Katarzyna P Adamala (Genetics, Cell Biology and Development, University of Minnesota), Aaron E Engelhart (Genetics, Cell Biology and Development, University of Minnesota)
Abstract:
The SARS-CoV-2 pandemic has highlighted the importance of early, rapid and widespread pathogen detection tests that are easily accessible and available to all. We have developed Apta-NASBA, an isothermal, cell-free, synthetic biology-based detection system that combines Nucleic Acid Sequence Based Amplification (NASBA) with transcription of fluorescent aptamers. Despite using the highly active and promiscuous T7 polymerase in the system, 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. With a limit of detection of 1 pM for the E. coli AggR pathogenic template, Apta-NASBA is highly sensitive and can yield multiplexed, multicolor, real-time fluorescent readout. 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 employing a cell phone camera module. This method is compatible with field detection and has potential for use in point-of-care SARS-CoV-2 diagnostics. We will discuss our work with Apta-NASBA and other isothermal amplification technologies.
References:
Aufdembrink LM, Khan P, Gaut NJ, Adamala KP, Engelhart AE. Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout. RNA. 2020 Sep;26(9):1283-1290. doi: 10.1261/rna.075192.120.
Keywords: NASBA, T7 RNA polymerase, Isothermal amplification