Rapid, adaptable and sensitive Cas13-based COVID-19 diagnostics using ADESSO
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Abstract
During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants.
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SciScore for 10.1101/2021.06.17.21258371: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Improve Medical Instruments, Guanzhou/China) and washed out with 2 ml 0,9% saline within 12 h of collection. Sex as a biological variable not detected. Randomization not detected. Blinding RNA extraction: For the first blind test (Figure 1), RNA was extracted from the clinical samples with the QIAamp® Viral RNA Mini kit (Qiagen, #52904) following the manufacturer’s instructions (140μl of swab were extracted and eluted in 60μl). Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources Cas13 purification: Plasmid encoding LwaCas13 (pC013 - Twinstrep-SUMO-huLwCas13a was a gift from Feng Zhang (Addgene plasmid # 90097; http://n2t.net/addgene:90097; … SciScore for 10.1101/2021.06.17.21258371: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: Improve Medical Instruments, Guanzhou/China) and washed out with 2 ml 0,9% saline within 12 h of collection. Sex as a biological variable not detected. Randomization not detected. Blinding RNA extraction: For the first blind test (Figure 1), RNA was extracted from the clinical samples with the QIAamp® Viral RNA Mini kit (Qiagen, #52904) following the manufacturer’s instructions (140μl of swab were extracted and eluted in 60μl). Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources Cas13 purification: Plasmid encoding LwaCas13 (pC013 - Twinstrep-SUMO-huLwCas13a was a gift from Feng Zhang (Addgene plasmid # 90097; http://n2t.net/addgene:90097; RRID:Addgene_90097)39 was transformed into Rosetta cells and purified according to established protocols with substantial modification. detected: RRID:Addgene_90097)Software and Algorithms Sentences Resources Once the whole reaction volume was absorbed, the dipstick was removed and photographed with a smartphone camera for band intensity quantification performed with the freely available ImageJ image processing program77. ImageJsuggested: (ImageJ, RRID:SCR_003070)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Rapid PCR and antigen-based tests are also available, but there are some limitations for their widespread use, such as the requirement for sophisticated PCR equipment and the standard practice of confirming positive antigen-based test results by RT-qPCR. Therefore, an alternative test that overcomes these limitations is still needed. Here, we have optimised the Cas13a-based diagnostic platform called SHERLOCK39 and developed the improved protocol ADESSO for highly sensitive COVID-19 testing. Overall, we tested 983 samples (496 positive and 487 negative, Supplementary File 1), in parallel comparison with RT-qPCR. To our knowledge, it is the first time that such an extensive study on clinical samples has been reported for CRISPR Dx technologies. ADESSO has a sensitivity of 96% on RNA extracted from swabs and a sensitivity of 77% when performed directly on unextracted swab samples. This drop in sensitivity is due to a decreased LoD at Ct 29, corresponding to low viral titers and minimal infectiousness11,51. However, skipping the RNA extraction step considerably reduces the sample-to-result turnaround time and allows more frequent testing, which is suggested to be essential for efficient identification of viral infections and isolation of carriers to contain the pandemic30. Other advantages are the lower need for RNA extraction kits, whose shortage has been a global issue throughout the pandemic57,58, and the higher test portability, which makes ADESSO appropriate for POC testing...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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