Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay
Open Access
- 30 September 2021
- journal article
- research article
- Published by Life Science Alliance, LLC in Life Science Alliance
- Vol. 4 (12), e202101213
- https://doi.org/10.26508/lsa.202101213
Abstract
The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA–based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.Funding Information
- Azim Premji Philanthropic initiative, Punjab National Bank Housing, and IQVIA
- Department of Atomic Energy, Government of India and the National Centre for Biological Sciences-TIFR (12-R&D-TFR-5.04-0800)
This publication has 63 references indexed in Scilit:
- Engineering Insights for Multiplexed Real-Time Nucleic Acid Sequence-Based Amplification (NASBA): Implications for Design of Point-of-Care DiagnosticsMolecular Diagnosis & Therapy, 2013
- Fiji: an open-source platform for biological-image analysisNature Methods, 2012
- Primer3—new capabilities and interfacesNucleic Acids Research, 2012
- NUPACK: Analysis and design of nucleic acid systemsJournal of Computational Chemistry, 2010
- NUCLEIC ACID SEQUENCE‐BASED AMPLIFICATION (NASBA) IN MOLECULAR BACTERIOLOGY: A PROCEDURAL GUIDEJournal of Rapid Methods and Automation in Microbiology, 2007
- Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)Molecular Biotechnology, 2002
- Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood SamplesJournal of Clinical Microbiology, 2001
- Evaluation of β-Galactosidase Activity in Tissue in the Presence of BloodJournal of Vascular Research, 2000
- A novel quantitative multiplex NASBA method: application to measuring tissue factor and CD14 mRNA levels in human monocytesNucleic Acids Research, 1999
- Nucleic acid sequence-based amplificationNature, 1991