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An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12

Lucía A. CurtiiD, Federico Pereyra-Bonnet, Carla Alejandra GiméneziD

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received global attention due to the recent outbreak in China. In this work, we report a CRISPR-Cas12 based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. These key traits of the CRISPR method are critical for virus detection in regions that lack resources to use the currently available methods.

In-depth summary

Claude Opus 5.5 · max effort · 8 Nov 2026 · from the full text of version 1 (2 Mar 2020) · CC BY-NC-ND 4.0

Reverse-transcription recombinase polymerase amplification (RT-RPA) plus LbCas12a collateral cleavage detected synthetic SARS-CoV-2 ORF1ab RNA down to 10 copies/µL by fluorescence, with similar lateral-flow strip sensitivity.

The SARS-CoV-2 detection protocols compiled by the WHO all rely on real-time reverse transcription PCR (rRT-PCR), which demands specialized staff and costly instruments often missing in low-resource regions. This work tests whether Cas12a collateral cleavage of single-stranded DNA (ssDNA) reporters, read by fluorescence or paper lateral-flow strips, can offer a sensitive, portable assay at a stated cost of USD 1-2 per reaction.

Key results

  1. ShownFluorescence readout of the RT-RPA plus LbCas12a assay detected synthetic SARS-CoV-2 ORF1ab RNA with an estimated limit of detection (LOD) of 10 copies/µL, which the authors place below clinically reported viral loads.

    LOD read from background-subtracted fluorescence (sample minus no-target control) at the 30 min reading, mean ± SD (n=3); 10 copies/µL is stated to be 4 orders of magnitude below the 10^5 copies/µL viral load reported for a patient in Berlin. · Results and Discussion; Figure legends

  2. SuggestedReplacing the FAM reporter with a biotin-labeled ssDNA reporter read on commercial lateral-flow paper strips gave a detection limit the authors describe as similar to the plate-reader assay, enabling readout without a fluorimeter.

    On Milenia HybriDetect 1 strips with target in buffer (Figure 2B), the authors report an LOD similar to the fluorescence format with no loss of sensitivity; the text gives no separate numeric LOD for the strip format. · Materials and methods; Results and Discussion; Figure legends

  3. SuggestedSynthetic SARS-CoV-2 RNA spiked into pretreated saliva from a healthy donor still yielded a positive strip, which the authors interpret as no inhibition of the reaction by saliva constituents.

    Saliva spiked at 10^5 copies/µL was heat- and chemically treated, and 2 µL served as RT-RPA input; positive strip in Figure 2C. The legend says saliva optimization continues to obtain a stronger strip signal; no other spike levels or patient saliva are reported. · Materials and methods; Results and Discussion; Figure legends

  4. ClaimedThe workflow is presented as rapid and inexpensive: under 60 min for both detection strategies schematized in Figure 1, at USD 1-2 per reaction.

    The Figure 1 legend states both strategies take less than 60 min and the Conclusions cite USD 1-2/reaction, without timed end-to-end runs or a cost breakdown; Methods specify a 30 min RT-RPA at 42°C and detection incubations of up to 90 min at 37°C. · Materials and methods; Conclusions; Figure legends

  5. ClaimedAmong the RdRp, ORF1b and ORF1ab target regions tested, ORF1ab amplified with China CDC-suggested primers gave the best detection performance.

    The authors state the best results came from ORF1ab with China CDC primers, but the text reports no comparative LOD or signal data for the RdRp or ORF1b targets. · Materials and methods; Results and Discussion

Methods, data and reproducibility

Synthetic SARS-CoV-2 RNA fragments (RdRp, ORF1b, ORF1ab) were amplified by one-step RT-RPA and detected through collateral cleavage of FAM or biotin ssDNA reporters by an LbCas12a-single guide RNA (sgRNA) complex, read on a fluorescence plate reader or lateral-flow strips; spiked healthy-donor saliva served as a mock clinical sample.

  • Synthetic SARS-CoV-2 RNA fragments of the RdRp, ORF1b and ORF1ab regions, designed from the WH-human1 reference and produced by fill-in PCR plus T7 in vitro transcription · SARS-CoV-2 (synthetic RNA) · n = n=3 per data point (Figure 2) · GenBank MN908947
  • Saliva from a healthy donor spiked with synthetic SARS-CoV-2 RNA (10^5 copies/µL), heat- and chemically pretreated, 2 µL used as RT-RPA input to mimic a clinical specimen · human (saliva matrix) with synthetic SARS-CoV-2 RNA · n = a single healthy donor; sample count not stated
Methods & tools
LbCas12a endonuclease (NEB) with in-house in vitro transcribed sgRNA, TwistAmp Basic recombinase polymerase amplification (RPA) kit (TwistDx), M-MuLV reverse transcriptase and murine RNase inhibitor (NEB), NEBNext High-Fidelity 2X PCR Master Mix (fill-in PCR), In vitro transcription kit (Ambion, Invitrogen) with T7 promoter templates, NEBuffer 2.1 (NEB), FAM- and biotin-labeled custom ssDNA reporters, SpectraMax M2 fluorescence plate reader (Molecular Devices), 384-well format, Milenia HybriDetect 1 lateral flow system (TwistDx), China CDC-suggested ORF1ab primer pair, Microsoft Excel 2016, GraphPad Prism v.8.1.2
Reagents & environment
Microsoft Excel 2016 for data arrangement; GraphPad Prism v.8.1.2 for plotting.
Reproducibility gaps
No data or code availability statement; sequences of targets, sgRNA, RT-RPA primers and reporters are deferred to Table 1, whose contents are absent from the text; saliva heat and chemical pretreatment conditions are unspecified.
Limitations (4)
  • Only synthetic RNA was assayed; no patient specimens were tested because, per the authors, no clinical cases had yet been reported in their region (South America), so the work is framed as proof-of-principle. · stated by the authors
  • Saliva performance remains preliminary: the authors note that conditions in saliva are still being optimized to obtain a more intense paper-strip signal. · stated by the authors
  • No specificity data are reported: no cross-reactivity testing against SARS-CoV (noted as having high sequence identity), other coronaviruses or respiratory pathogens, and no negative clinical samples, so false-positive rates are unknown. · noted in this summary
  • The LOD rests on triplicate background-subtracted fluorescence at one 30 min reading, with no stated positivity threshold, replicate hit-rate analysis or head-to-head rRT-PCR comparison; saliva was spiked only at 10^5 copies/µL, leaving its matrix LOD undefined. · noted in this summary

Read this if you are setting up an RT-RPA plus Cas12a lateral-flow test for SARS-CoV-2 and want early reagent conditions and a synthetic-RNA sensitivity estimate, not clinical validation.

Citations
150

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