PACRAT: Pathogen detection with aptamer-detected cascaded recombinase polymerase amplification-in vitro transcription

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods employ the recombinase polymerase amplification (RPA), which exhibits PCR-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (Pathogen detection with Aptamer-detected Cascaded Recombinase polymerase Amplification-in vitro Transcription) can be used to detect SARS-CoV-2 with single-copy detection limits and 10-minute detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be employed for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.