Detection of viral RNAs at ambient temperature via reporter proteins produced through the target-splinted ligation of DNA probes

Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection. We used INSPECTR to detect a panel of five respiratory viral targets in a single reaction via a lateral-flow readout and similar to 4,000 copies of viral RNA via additional ambient-temperature rolling circle amplification of the expression cassette. Leveraging synthetic biology to simplify workflows for nucleic acid diagnostics may facilitate their broader applicability at the point of care.

Automated summary

This study presents a cost-effective and simplified nucleic acid detection technique, named INSPECTR, which enables accurate and multiplexed detection at ambient temperature. It utilizes target-specific DNA probes and flexible expression cassettes to generate reporter proteins for linear detection and visual detection. Leveraging synthetic biology, this technique could potentially enhance the applicability of nucleic acid diagnostics at point-of-care settings.