Clinical validation of RCSMS: a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva
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Abstract
Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the “gold standard” method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Perú (current status over a total of 350 samples ). We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a “point of care” test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.
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SciScore for 10.1101/2021.04.26.21256081: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All the participants read and signed informed consents prior to providing samples for this study. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Amplification and detection by RT-qPCR: The RT-qPCR of viral genes Orf1ab and N run at UPCH used the in vitro diagnostic (IVD) FTD-SARS-CoV-2 kit (Siemens) and QTower 3G real-time thermocyclers (Analytik-Jena). UPCHsuggested: NoneAll analyses were performed with the Stata software version 16.0 (StataCorp, College Station, TX, USA). Statasuggested: (Stata, RRID:SCR_012763)StataCorpsuggested: (Stata, RRID:SCR_012763)For… SciScore for 10.1101/2021.04.26.21256081: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: All the participants read and signed informed consents prior to providing samples for this study. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Amplification and detection by RT-qPCR: The RT-qPCR of viral genes Orf1ab and N run at UPCH used the in vitro diagnostic (IVD) FTD-SARS-CoV-2 kit (Siemens) and QTower 3G real-time thermocyclers (Analytik-Jena). UPCHsuggested: NoneAll analyses were performed with the Stata software version 16.0 (StataCorp, College Station, TX, USA). Statasuggested: (Stata, RRID:SCR_012763)StataCorpsuggested: (Stata, RRID:SCR_012763)For data visualization and graphs GraphPad Prism 7.00 was used (GraphPad Software, La Jolla California USA, www.graphpad.com). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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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