Towards detection of SARS-CoV-2 RNA in human saliva: A paper-based cell-free toehold switch biosensor with a visual bioluminescent output.

Autor: Hunt JP; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Zhao EL; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Free TJ; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Soltani M; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Warr CA; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Benedict AB; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA., Takahashi MK; Department of Biology, California State University Northridge, Northridge, CA, USA., Griffitts JS; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA., Pitt WG; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA., Bundy BC; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA. Electronic address: bundy@byu.edu.
Jazyk: angličtina
Zdroj: New biotechnology [N Biotechnol] 2022 Jan 25; Vol. 66, pp. 53-60. Date of Electronic Publication: 2021 Sep 21.
DOI: 10.1016/j.nbt.2021.09.002
Abstrakt: The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 μL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson.
(Copyright © 2021. Published by Elsevier B.V.)
Databáze: MEDLINE
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