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Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test

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Abstract

The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30–45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants.

Key points

  • MARVE is a paper-based, preamplification-free assay for the detection of viral variants using toehold exchange (TE) DNA probes. The TEprobe binds to the target viral RNA and starts an enzymatic reaction, which results in low-pH conditions and a color change visible to the eye.

  • The high specificity of a nucleic acid test, combined with low cost and ease of use, makes MARVE suitable for home testing.

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Fig. 1: A schematic illustration of the MARVE assay detection process.
Fig. 2: An overview of the MARVE assay.
Fig. 3: Pseudotyped virus construction.
Fig. 4: The pipeline for designing and optimizing TEprobes.
Fig. 5: Origami paper preparation.
Fig. 6: Construction of the enclosure for the smartphone.
Fig. 7: Image processing for an origami paper.
Fig. 8: A mobile web app for COVID-19 self-testing.
Fig. 9: An operation graphic of manually folding origami papers for viral RNA detection.
Fig. 10: Anticipated results for the visual detection of coronaviruses.
Fig. 11: Detection of three coronaviruses (SARS-CoV-2, SARS-CoV and MERS-CoV) spiked into throat swabs, frozen belt fish and food packaging.
Fig. 12: SARS-CoV-2 variant detection.
Fig. 13: Diagnostics of SARS-CoV-2 and its variants in clinical samples.

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Data availability

The data supporting the findings of this study are available within the source data and the supporting primary research papers16,17. Source data are provided with this paper.

Code availability

The mobile web apps for COVID-19 self-testing and viral contamination monitoring in food can be visited at http://47.109.38.99/origami1/ or at http://47.109.38.99/origami2/, respectively. The code for designing TEprobes is available on GitHub at https://github.com/Nelson233/TEprobe-design. The code for the two web apps is provided on GitHub at https://github.com/Nelson233/origami1 and https://github.com/Nelson233/origami2, respectively. The code for image analysis using MATLAB software is available on GitHub at https://github.com/Nelson233/MARVEL.

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Acknowledgements

We thank all authors of the primary research paper. The work was funded by the National Key Research and Development Program of China (2023YFB3208302 and 2021YFA1200104), the New Cornerstone Investigator Program, National Natural Science Foundation of China (22074100 and 22027807), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB36000000) and Tsinghua-Vanke Special Fund for Public Health and Health Discipline Development (2022Z82WKJ003).

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Author notes

  1. These authors contributed equally: Ting Zhang, Yuxi Wang.

Authors and Affiliations

  1. Department of Chemistry, Center for BioAnalytical Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, New Cornerstone Science Institute, Tsinghua University, Beijing, China

    Ting Zhang, Xucong Teng & Jinghong Li

  2. College of Biomass Science and Engineering, Department of Respiration and Critical Care Medine, West China Hospital, Sichuan University, Chengdu, China

    Ting Zhang, Yuxi Wang & Ruijie Deng

  3. Beijing Institute of Life Science and Technology, Beijing, China

    Xucong Teng & Jinghong Li

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Contributions

J.L. and R.D. conceived and designed the protocol. R.D. and T.Z. developed the protocol. T.Z., R.D., Y.W. and X.T. performed the experiments and analyzed the data. R.D. and T.Z. wrote the manuscript. J.L. edited the manuscript. All authors read, commented on and accepted the final manuscript.

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Correspondence to Ruijie Deng or Jinghong Li.

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Nature Protocols thanks Changqing Yi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references that have used this protocol

Zhang, T. et al. Nat. Biomed. Eng. 6, 957–967 (2022): https://doi.org/10.1038/s41551-022-00907-0

Zhang, T. et al. Nat. Commun. 14, 4327 (2023): https://doi.org/10.1038/s41467-023-39952-x

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Tables 1 and 2.

Supplementary Video 1

The illustration of paper folding using the enclosure.

Supplementary Data 1

The original CAD file of eight-site origami paper.

Supplementary Data 2

The PDF file of eight-site origami paper.

Supplementary Data 3

The original CAD file of four-site origami paper

Supplementary Data 4

The PDF file of four-site origami paper.

Supplementary Data 5

The original file of the enclosure.

Supplementary Data 6

The original CAD file of 100-site origami paper.

Supplementary Data 7

The PDF file of 100-site origami paper.

Source data

Source Data Fig. 10

Statistical source data.

Source Data Fig. 11

Statistical source data.

Source Data Fig. 12

Statistical source data.

Source Data Fig. 13

Statistical source data.

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Zhang, T., Wang, Y., Teng, X. et al. Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01022-x

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