Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
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 α-complementation of β-galactosidase ω-fragment (LacZω) with cell-free expressed LacZα, using an X-gal analogue as a substrate. The estimated cost of single reaction is <€1/test, which may facilitate diagnostic kit accessibility in developing countries.
Article activity feed
-
SciScore for 10.1101/2021.11.29.21267025: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: The RNA from infected patients was derived from anonymized saliva samples collected with ethical committee approval of the Azienda Provinciale per i Servizi Sanitari of the Autonomous Province of Trento (P.A.T.). Sex as a biological variable not detected. Randomization Reverse Transcriptase 128 U (ThermoFisher), 32 U Hi®-T7 RNA polymerase (New England Biolabs), 500 nM random duplex primer pair, 1 µL of template RNA (varying amounts), 10% (v/v) DMSO, filled up to 10 µL of final volume with RNase/DNase-free water. Blinding not detected. Power Analysis not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources For on-paper reactions, the E. coli strains JM109 and … SciScore for 10.1101/2021.11.29.21267025: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: The RNA from infected patients was derived from anonymized saliva samples collected with ethical committee approval of the Azienda Provinciale per i Servizi Sanitari of the Autonomous Province of Trento (P.A.T.). Sex as a biological variable not detected. Randomization Reverse Transcriptase 128 U (ThermoFisher), 32 U Hi®-T7 RNA polymerase (New England Biolabs), 500 nM random duplex primer pair, 1 µL of template RNA (varying amounts), 10% (v/v) DMSO, filled up to 10 µL of final volume with RNase/DNase-free water. Blinding not detected. Power Analysis not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources For on-paper reactions, the E. coli strains JM109 and DH10β were used. JM109suggested: NoneRecombinant DNA Sentences Resources LacZω was cloned into a modified pMAL-c4X backbone containing N-terminus Maltose Binding Protein (MBP) with TEV protease recognition site flanked by (GS)2 linker sequence. pMAL-c4Xsuggested: RRID:Addgene_75288)The sequence was synthesized as a dsDNA gene fragment from GenScript and cloned into an expression cassette (in plasmid pSB1A3) under the control of consensus T7 promoter, with additional 5’ flanking ATT and 3’ flanking GG sequences. pSB1A3suggested: RRID:Addgene_116852)Software and Algorithms Sentences Resources qRT-PCR was performed by SsoAdvanced™ SsoAdvanced™suggested: NoneThe extract was separated from the glass beads by centrifugation at 6000 g, 4 °C for 10 min, with Bio–Rad Bio–Spin columns. Bio–Spinsuggested: NoneBio–Radsuggested: NoneThe extract was separated from the glass beads by centrifugation at 6000 g, 4 °C for 10 min, with Bio–Rad Bio–Spin columns. Bio–Spinsuggested: NoneBio–Radsuggested: NoneFinal reaction mixture contained 1x reaction buffer, deoxyribonucleotide mix (1 mM each), ribonucleotide mix (2 mM each), forward and reverse primers ([final] = 1 µM each), 0.01 U of Yeast inorganic pyrophosphatase (YIPP, New England Biolabs), 0.4 U RiboLock RNase inhibitor (ThermoFisher), 0.08 U RNaseH (New England Biolabs), RevertAid Reverse Transcriptase 128 U (ThermoFisher), 32 U Hi®-T7 RNA polymerase (New England Biolabs), 500 nM random duplex primer pair, 1 µL of template RNA (varying amounts), 10% (v/v) DMSO, filled up to 10 µL of final volume with RNase/DNase-free water. ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Stem-region melting temperatures were calculated according to Primer3 software defaults (Santa Lucia 1998). Primer3suggested: (Primer3, RRID:SCR_003139)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a protocol registration statement.
Results from scite Reference Check: We found no unreliable references.
-
