Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

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 employing a cell phone camera module, compatible with field detection.

T7Max transcription system

“…[30] Apta-NASBA is an isothermal exponential disease detection reaction, dependent on the productivity of T7 RNA polymerase. [31] In Apta-NASBA, primers introduce the T7 RNA polymerase promoter and result in a fluorescent read out via an RNA aptamer. We designed primers to detect the aggR gene of E. Colione incorporating T7Max and the other, classic T7.…”

“…To prepare the reverse transcription reaction, 2 μL of the DNase-treated sample was mixed with 2 μ L of Apta-NASBA reactions Apta-NASBA reactions were performed as previously described. [31] Primers used for the Apta-NASBA reaction were: Broccoli aptamer coding primer (broccoli is in italics) 5'-GAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCAGCGATACATTAAGACGCCTAAAG-3' classic T7 primer (promoter is in italics) 5'-TAATACGACTCACTATAGCGTCAGCATCAGCTACAATTATTCC-3' T7Max primer (promoter is in italics) 5'-…”

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings

“…Unrau and coworkers recently demonstrated the use of the Mango aptamer in Nested Mango NASBA, 79 , 80 and Engelhart and coworkers showed multiplexed detection with cell phone camera-based readout using the Broccoli, Corn, and malachite green aptamers in Apta-NASBA. 81 These techniques enable an inexpensive fluorogenic real-time readout. NASBA has also been detected by an aptamer-based molecular beacon approach.…”

“…NASBA has the capability of starting with either DNA or RNA, with RNA as the main reaction product, enabling the use of functional nucleic acids such as aptamers and ribozymes in the reaction product, opening the door to using a variety of detectable signals. 79 , 81 By applying fluorescent RNA aptamers for the readout, it is possible to obtain comparable specificity as a fluorescent probe, but with the cost of an intercalating dye such as SYBR Green. The optimal reaction temperature is 37–42 °C, lower than that of LAMP or RPA.…”

“…used a competitor DNA duplex to do so. 79 , 81 As with other multienzyme isothermal reactions, the use of multiple enzymes necessarily adds complexity to manufacturing processes.…”