Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the -complementation of {beta}-galactosidase {omega}-fragment (LacZ{omega}) with cell-free expressed LacZ, using an X-gal analogue as a substrate. The estimated cost of single reaction is <{euro}1/test, which may facilitate diagnostic kit accessibility in developing countries.