Background:The current outbreak of SARS-CoV-2 has spread to almost every country with more than three million confirmed cases and over two hundred thousand deaths as of April 28, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. Methods: We used computational and manual design to generate a suitable set of RT-RPA primer and exo-IQ probe sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated by means of a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. Results: The 95 % detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87 -27.39) RNA copies per reaction. The assay showed no crossreactivity to any other screened coronaviruses as well as respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n=20). Conclusions: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple to use alternative to RT-qPCR for first-line screening at the point of need. Downloaded from https://academic.oup.com/clinchem/advance-article-abstract/doi/10.1093/clinchem/hvaa116/5834714 by guest on 09 June 2020

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

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The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.