Nearly 60% of people infected with SARS-CoV-2 develop mild symptoms or no symptoms¹. There was an urgent need for the development of fast, simple and high-throughput nucleic acid detection to solve the problem that RT-qPCR might not have the ability to deal well with the serious challenge for global public health security.

We herein presented a novel amplification-free rapid SARS-CoV-2 nucleic acid detection platform based on hybrid capture fluorescence immunoassay (HC-FIA). The application of the S9.6 monoclonal antibody underlies the design of our method, which recognizing DNA-RNA double-stranded hybrids and enabling the conversion of nucleic acid testing into immunofluorescence carrying on a simple lateral flow dipstick (Figure 1).

The whole test procedure involved two steps, namely hybridization and immunofluorescence analysis, and can be finished in less than an hour. Optimized DNA probes targeting the conserved open reading frame 1ab (ORF1ab), envelope protein (E) and the nucleocapsid (N) regions of the SARS-CoV-2 genome were documented to have limit of detection (LOD) of 500 copies/mL for clinical throat swab samples, comparable to the commercial kits based on RT-qPCR. And no significant cross-reaction was found between the SARS-CoV-2 probes and 55 common pathogens. The HC-FIA assay is also documented to be robust to interference such as hemoglobin, mucin and various possible drugs that might exist in clinical samples. The assay and test kit could be adapted for the detection of other viral RNA.

Being supported by a multi-hospital randomized double-blind trial involving 734 samples, the HC-FIA presented herein was not only a proof-of-concept research, but also had been developed into a commercial test kit for the diagnosis of SARS-CoV-2, which recently approved by the National Medical Products Administration (NMPA) and acquired the European Conformity certification.

SARS-CoV-2 can be inactivated by incubation at 56 °C for 30 min, and the absence of nucleic acid amplification means that the assay does not suffer from contamination by amplicons. The materials needed for each test cost as little as US$2. It relies little on equipment and professional operator, realizing confirming infection in a "suitcase laboratory”. It can be developed into on-site detection method for outpatient department, emergency department, customs and grassroots disease control.

CRISPR-based assays on the basis of Cas13 or Cas12 endonucleases have also been developed using lateral flow technology^2-6. However, these assays require nucleic acid extraction and recombinase polymerase amplification or loop-mediated isothermal amplification. Assay times of CRISPR-based diagnostics and HC-FIA for the detection of SARS-CoV-2 are similar, yet the LOD values of the CRISPR-based diagnostics are higher (in the order of 10⁴–10⁵ copies per ml for SARS-CoV-2 ^2,6 and 1,000 copies per ml for the detection of the Zika virus and the dengue virus⁴).

We are confident that the HC-FIA test was capable of serving as a sure safeguard in containing the epidemic through reducing community spread and imported cases from its root causes, especially in the developing countries.

References:

1. Qiu, J. Covert coronavirus infections could be seeding new outbreaks. Nature https://doi.org/10.1038/d41586-020-00822-x (2020).
2. Abudayyeh, O., Gootenberg, J. & Zhang, F. Enabling Coronavirus Detection Using CRISPR-Cas13: An Open-Access SHERLOCK Research Protocol (2020); https://mcgovern.mit.edu/2020/02/14/enabling-coronavirus-detection-using-crispr-cas13-an-open-access-sherlock-research-protocol/
3. Myhrvold, C., Freije, C. A. & Gootenberg, J. S. Field-deployable viral diagnostics using CRISPR-Cas13. Science 360, 444–448 (2018).
4. Freije, C. A. et al. Programmable inhibition and detection of RNA viruses using Cas13. Mol. Cell 76, 826–837 (2019).
5. Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O. & Zhang, F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat. Protoc. 14, 2986–3012 (2019).
6. Broughton., J. P. et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0513-4 (2020).